Transcriptional Regulation via AR and β3-AdR

HEK293FT cells were seeded in 48-well plates at 6.0 × 10⁴ cells/well using steroid-free medium and cultured overnight. For determination of CRE- or androren response element (ARE)-mediated transcriptional activity, cells were transfected with β3-AdR expression vector (pcDNA3.2-β3-AdR-V5 (74 (link))), AR expression vector (pcDNA3.1-AR (68 (link)), pcDNA3.1-AR (K630A/K632A/K633A), pcDNA3.1-AR (C619Y) (32 (link), 75 (link)), pcDNA3.1-AR (L26A/F27A) (76 (link)), pcDNA3.1-AR (E897Q) (76 (link)), pcDNA3.1-AR (ΔpolyQ+Q6+Q5) (35 (link)), pcDNA3.1-AR (C806A)), luciferase reporter vector (p4xCRE-TATA-Luc2P (74 (link)), pGL4-ARE2-TATA-Luc (77 (link)), or pUCP1-pro-Luc2P), and Renilla luciferase reporter vector (pGL4.74[hRluc/TK] (Promega)) with PEI MAX (Polysciences Inc) and Opti-MEM (Thermo Fisher Scientific) for 24 h. For the mammalian two-hybrid assay, cells were transfected with pGAL-CREB, pcDNA3.1-AR or pACT-AR (NTD) (76 (link)), pG5luc (Promega), and pGL4.74[hRluc/TK] with PEI MAX and Opti-MEM for 24 h. Cells were incubated in the presence of 10 nM DHT for 16 h and then stimulated with 10 μM CL316243 or 10 μM forskolin for an additional 4 h. Cells were treated with the AR antagonist bicalutamide (10 μM) 30 min prior to DHT treatment. Luciferase reporter activity was determined as described previously (68 (link)). Data are expressed as relative light units.

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Harada N., Kubo K., Onishi T., Kitakaze T., Goto T., Inui H, & Yamaji R. (2022). Androgen receptor suppresses β-adrenoceptor-mediated CREB activation and thermogenesis in brown adipose tissue of male mice. The Journal of Biological Chemistry, 298(12), 102619.

Publication 2022

pGL4.74[hRluc/TK] PEI MAX Opti-MEM pG5luc

Bicalutamide Cells Cl316243 Dht nm Forskolin Light Luciferase Mammalian Pgal Pgl4 Promega Renilla luciferase Response element Steroid Transcriptional Two hybrid assay Vector

Corresponding Organization :

Other organizations : Osaka Prefecture University, Kyoto University, Otemae University

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Variable analysis

independent variables

β3-AdR expression vector (pcDNA3.2-β3-AdR-V5)
AR expression vector (pcDNA3.1-AR, pcDNA3.1-AR (K630A/K632A/K633A), pcDNA3.1-AR (C619Y), pcDNA3.1-AR (L26A/F27A), pcDNA3.1-AR (E897Q), pcDNA3.1-AR (ΔpolyQ+Q6+Q5), pcDNA3.1-AR (C806A))
Luciferase reporter vector (p4xCRE-TATA-Luc2P, pGL4-ARE2-TATA-Luc, pUCP1-pro-Luc2P)
Renilla luciferase reporter vector (pGL4.74[hRluc/TK])
PGAL-CREB
PcDNA3.1-AR or pACT-AR (NTD)
PG5luc
10 nM DHT
10 μM CL316243
10 μM forskolin
10 μM bicalutamide

dependent variables

CRE- or androren response element (ARE)-mediated transcriptional activity
Luciferase reporter activity

control variables

HEK293FT cells seeded at 6.0 × 10^4 cells/well
Cells cultured overnight in steroid-free medium
Transfection using PEI MAX and Opti-MEM for 24 h
Cells incubated with 10 nM DHT for 16 h
Cells stimulated with 10 μM CL316243 or 10 μM forskolin for an additional 4 h
Cells treated with 10 μM bicalutamide 30 min prior to DHT treatment

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