Limits...
IRE1α-XBP1 is a novel branch in the transcriptional regulation of Ucp1 in brown adipocytes.

Asada R, Kanemoto S, Matsuhisa K, Hino K, Cui M, Cui X, Kaneko M, Imaizumi K - Sci Rep (2015)

Bottom Line: We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1.Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression.These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Institute of Biomedical &Health Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

ABSTRACT
The unfolded protein response (UPR) not only resolves endoplasmic reticulum (ER) stress, but also regulates cellular physiological functions. In this study, we first linked the UPR to the physiological roles of brown adipose tissue (BAT). BAT is one of the tissues that control energy homeostasis in the body. Brown adipocytes are able to dissipate energy in the form of heat owing to their mitochondrial protein, uncoupling protein 1 (UCP1). We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1. Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression. However, the activation of the IRE1α-XBP1 pathway by ER stress never upregulated Ucp1. On the other hand, the activation of protein kinase A (PKA) induced Ucp1 transcription through the activation of IRE1α-XBP1. The inhibition of PKA abrogated the activation of IRE1α-XBP1 pathway, while the inhibition of a p38 mitogen activated protein kinase (p38 MAPK), which is one of the downstream molecules of PKA, never suppressed the activation of IRE1α-XBP1 pathway. These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

No MeSH data available.


Related in: MedlinePlus

The IRE1α-XBP1 pathway plays a crucial role in the transcriptional induction of Ucp1.(a) RT-PCR analysis of Xbp1 in brown adipocytes that were pre-treated with 30 μM 4 μ8 C for 30 min and then stimulated with 1 μM CL 316,243 for 3 h (upper panel). Lower graph is the quantification of Xbp1 splicing levels. (b) Real-time PCR analysis of Ucp1 in brown adipocytes treated with 4 μ8 C and CL 316,243 described as (a). Note that treatment with 4 μ8 C significantly decreased Ucp1 expression induced by CL 316,243. Data are mean ± S.D. (n = 5), **P < 0.01, ***P < 0.001. (c) RT-PCR analysis of Xbp1 in brown adipocytes that were treated for 3 h with tunicamycin (Tm) at the indicated concentrations (upper panel). Lower graph shows the quantification of Xbp1 splicing levels. (d) Real-time PCR analysis of Ucp1 in brown adipocytes treated with Tm described as (c). Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, cont.: control. (e) Luciferase assay using C3H10T1/2 cells that were transfected with vectors expressing sXBP1 or ΔbZIP-sXBP1, and then treated with 20 μM forskolin (FSK) for 8 h. Note that the increase in the reporter activity by sXBP1 expression and FSK treatment was higher than the activity by FSK treatment alone. Data are mean ± S.D. (n = 6), *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4644985&req=5

f3: The IRE1α-XBP1 pathway plays a crucial role in the transcriptional induction of Ucp1.(a) RT-PCR analysis of Xbp1 in brown adipocytes that were pre-treated with 30 μM 4 μ8 C for 30 min and then stimulated with 1 μM CL 316,243 for 3 h (upper panel). Lower graph is the quantification of Xbp1 splicing levels. (b) Real-time PCR analysis of Ucp1 in brown adipocytes treated with 4 μ8 C and CL 316,243 described as (a). Note that treatment with 4 μ8 C significantly decreased Ucp1 expression induced by CL 316,243. Data are mean ± S.D. (n = 5), **P < 0.01, ***P < 0.001. (c) RT-PCR analysis of Xbp1 in brown adipocytes that were treated for 3 h with tunicamycin (Tm) at the indicated concentrations (upper panel). Lower graph shows the quantification of Xbp1 splicing levels. (d) Real-time PCR analysis of Ucp1 in brown adipocytes treated with Tm described as (c). Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, cont.: control. (e) Luciferase assay using C3H10T1/2 cells that were transfected with vectors expressing sXBP1 or ΔbZIP-sXBP1, and then treated with 20 μM forskolin (FSK) for 8 h. Note that the increase in the reporter activity by sXBP1 expression and FSK treatment was higher than the activity by FSK treatment alone. Data are mean ± S.D. (n = 6), *P < 0.05, ***P < 0.001.

Mentions: To determine whether the IRE1α-XBP1 pathway is mainly involved in the transcriptional induction of Ucp1, we first attempted to investigate the effects of the knockdown of Xbp1 in brown adipocytes using lentiviral vectors, because primary mature brown adipocytes do not proliferate. However, we could not detect green fluorescent protein (GFP) fluorescence in primary mature brown adipocytes infected with lentiviruses expressing GFP, indicating that the knockdown of Xbp1 in mature brown adipocytes is technically difficult. Instead of gene silencing experiments, we treated primary brown adipocytes with 30 μM 4 μ8 C. 4 μ8 C inhibits the nuclease activity of IRE1α by directly binding to its active site without affecting its kinase activity or its overall dimerization or oligomerization states35. Using this compound, we successfully depleted the splicing of Xbp1 mRNA induced by the β3-AR pathway (Fig. 3a). The quantification of Ucp1 mRNA levels demonstrated a significant decrease after treatment with 4 μ8 C compared with treatment with CL 316,243 (Fig. 3b). Treatment with 4 μ8 C also inhibited the splicing of Xbp1 mRNA and the upregulation of Ucp1 expression following FSK treatment (Supplementary Fig. 3a,b), suggesting that the IRE1α-XBP1 pathway plays a crucial role in the transcriptional induction of Ucp1. Because ER stress activates the IREα-XBP1 pathway, we next examined whether ER stress is able to induce the transcription of Ucp1. Primary brown adipocytes were treated with various concentrations of tunicamycin (Tm), which blocks N-linked glycosylation and causes ER stress. Tm treatment caused significant splicing of Xbp1 mRNA (Fig. 3c), but never induced Ucp1 transcription (Fig. 3d). Therefore, the activation of the IRE1α-XBP1 pathway by the β3-AR pathway, which leads to the transcriptional induction of Ucp1, could occur independently of the canonical UPR that is induced by ER stress.


IRE1α-XBP1 is a novel branch in the transcriptional regulation of Ucp1 in brown adipocytes.

Asada R, Kanemoto S, Matsuhisa K, Hino K, Cui M, Cui X, Kaneko M, Imaizumi K - Sci Rep (2015)

The IRE1α-XBP1 pathway plays a crucial role in the transcriptional induction of Ucp1.(a) RT-PCR analysis of Xbp1 in brown adipocytes that were pre-treated with 30 μM 4 μ8 C for 30 min and then stimulated with 1 μM CL 316,243 for 3 h (upper panel). Lower graph is the quantification of Xbp1 splicing levels. (b) Real-time PCR analysis of Ucp1 in brown adipocytes treated with 4 μ8 C and CL 316,243 described as (a). Note that treatment with 4 μ8 C significantly decreased Ucp1 expression induced by CL 316,243. Data are mean ± S.D. (n = 5), **P < 0.01, ***P < 0.001. (c) RT-PCR analysis of Xbp1 in brown adipocytes that were treated for 3 h with tunicamycin (Tm) at the indicated concentrations (upper panel). Lower graph shows the quantification of Xbp1 splicing levels. (d) Real-time PCR analysis of Ucp1 in brown adipocytes treated with Tm described as (c). Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, cont.: control. (e) Luciferase assay using C3H10T1/2 cells that were transfected with vectors expressing sXBP1 or ΔbZIP-sXBP1, and then treated with 20 μM forskolin (FSK) for 8 h. Note that the increase in the reporter activity by sXBP1 expression and FSK treatment was higher than the activity by FSK treatment alone. Data are mean ± S.D. (n = 6), *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644985&req=5

f3: The IRE1α-XBP1 pathway plays a crucial role in the transcriptional induction of Ucp1.(a) RT-PCR analysis of Xbp1 in brown adipocytes that were pre-treated with 30 μM 4 μ8 C for 30 min and then stimulated with 1 μM CL 316,243 for 3 h (upper panel). Lower graph is the quantification of Xbp1 splicing levels. (b) Real-time PCR analysis of Ucp1 in brown adipocytes treated with 4 μ8 C and CL 316,243 described as (a). Note that treatment with 4 μ8 C significantly decreased Ucp1 expression induced by CL 316,243. Data are mean ± S.D. (n = 5), **P < 0.01, ***P < 0.001. (c) RT-PCR analysis of Xbp1 in brown adipocytes that were treated for 3 h with tunicamycin (Tm) at the indicated concentrations (upper panel). Lower graph shows the quantification of Xbp1 splicing levels. (d) Real-time PCR analysis of Ucp1 in brown adipocytes treated with Tm described as (c). Differences with and without treatment were analyzed by Student’s t-test. Data are mean ± S.D. (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, cont.: control. (e) Luciferase assay using C3H10T1/2 cells that were transfected with vectors expressing sXBP1 or ΔbZIP-sXBP1, and then treated with 20 μM forskolin (FSK) for 8 h. Note that the increase in the reporter activity by sXBP1 expression and FSK treatment was higher than the activity by FSK treatment alone. Data are mean ± S.D. (n = 6), *P < 0.05, ***P < 0.001.
Mentions: To determine whether the IRE1α-XBP1 pathway is mainly involved in the transcriptional induction of Ucp1, we first attempted to investigate the effects of the knockdown of Xbp1 in brown adipocytes using lentiviral vectors, because primary mature brown adipocytes do not proliferate. However, we could not detect green fluorescent protein (GFP) fluorescence in primary mature brown adipocytes infected with lentiviruses expressing GFP, indicating that the knockdown of Xbp1 in mature brown adipocytes is technically difficult. Instead of gene silencing experiments, we treated primary brown adipocytes with 30 μM 4 μ8 C. 4 μ8 C inhibits the nuclease activity of IRE1α by directly binding to its active site without affecting its kinase activity or its overall dimerization or oligomerization states35. Using this compound, we successfully depleted the splicing of Xbp1 mRNA induced by the β3-AR pathway (Fig. 3a). The quantification of Ucp1 mRNA levels demonstrated a significant decrease after treatment with 4 μ8 C compared with treatment with CL 316,243 (Fig. 3b). Treatment with 4 μ8 C also inhibited the splicing of Xbp1 mRNA and the upregulation of Ucp1 expression following FSK treatment (Supplementary Fig. 3a,b), suggesting that the IRE1α-XBP1 pathway plays a crucial role in the transcriptional induction of Ucp1. Because ER stress activates the IREα-XBP1 pathway, we next examined whether ER stress is able to induce the transcription of Ucp1. Primary brown adipocytes were treated with various concentrations of tunicamycin (Tm), which blocks N-linked glycosylation and causes ER stress. Tm treatment caused significant splicing of Xbp1 mRNA (Fig. 3c), but never induced Ucp1 transcription (Fig. 3d). Therefore, the activation of the IRE1α-XBP1 pathway by the β3-AR pathway, which leads to the transcriptional induction of Ucp1, could occur independently of the canonical UPR that is induced by ER stress.

Bottom Line: We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1.Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression.These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Institute of Biomedical &Health Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

ABSTRACT
The unfolded protein response (UPR) not only resolves endoplasmic reticulum (ER) stress, but also regulates cellular physiological functions. In this study, we first linked the UPR to the physiological roles of brown adipose tissue (BAT). BAT is one of the tissues that control energy homeostasis in the body. Brown adipocytes are able to dissipate energy in the form of heat owing to their mitochondrial protein, uncoupling protein 1 (UCP1). We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1. Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression. However, the activation of the IRE1α-XBP1 pathway by ER stress never upregulated Ucp1. On the other hand, the activation of protein kinase A (PKA) induced Ucp1 transcription through the activation of IRE1α-XBP1. The inhibition of PKA abrogated the activation of IRE1α-XBP1 pathway, while the inhibition of a p38 mitogen activated protein kinase (p38 MAPK), which is one of the downstream molecules of PKA, never suppressed the activation of IRE1α-XBP1 pathway. These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

No MeSH data available.


Related in: MedlinePlus