Fig. 1.
Design and characterization of the synthetic mammalian bile acid sensor. (A) Design of a synthetic bile acid-triggered signalling cascade. The G protein-coupled bile acid receptor 1 (TGR5) senses extracellular bile acids and triggers Gαs-mediated activation of adenylate cyclase, which converts ATP to cyclic AMP (cAMP). cAMP binds the regulatory subunits of protein kinase A (PKA), whose catalytic subunits translocate into the nucleus where they phosphorylate the cAMP-responsive binding protein 1 (CREB1). Subsequently, CREB1 binds and activates target gene transcription from synthetic promoters PCRE engineered to contain different CREB1 response elements (CRE). (B) Cholic acid-inducible transgene expression in HEK-293 cells using different TGR5 expression vectors. HEK-293 cells were co-transfected with pCK53 (PCRE-SEAP-pA; 100 ng) and either pTGR5 (PhCMV-TGR5-pA; 1000 ng) or pPB2 (PhCMV∗-1-TGR5-pA; 1000 ng) and cultivated in the presence or absence CA (100 μM). HEK-293 cells transfected with pSEAP2-Control (PSV40-SEAP-pA; 100 ng) were used as control. SEAP levels in the culture supernatants were scored after 24 h. Data presented are mean ± SD, n ⩾3. (C) SEAP expression kinetics. HEK-293 cells were co-transfected with pPB2 (PhCMV∗-1-TGR5-pA; 1000 ng) and either pCK53 (PCRE-SEAP-pA; 100 ng) or pSP16 (PCREm-SEAP-pA; 100 ng) and cultivated for 72 h in the presence or absence of CA (100 μM). SEAP levels in the culture supernatants were scored every 24 h. Data presented are mean ± SD, n ⩾3. (D) Dose-dependent SEAP expression. HEK-293 cells were co-transfected with pPB2 (PhCMV∗-1-TGR5-pA; 1000 ng) and either pSP16 (PCREm-SEAP-pA; 100 ng) or pCK53 (PCRE-SEAP-pA; 100 ng) and cultivated for 24 h in the presence of different CA concentrations (0–200 μM) before SEAP levels in the culture supernatants were scored. Data presented are mean ± SD, n ⩾3. (E) Bile acid-inducible SEAP expression in different mammalian cell lines. hMSC, Freestyle 293F, HEK-293, BHK-21, HT-1080 and CHO-K1 cells were co-transfected with pPB2/pSP16 (1000 ng/100 ng) and cultivated in medium containing different bile acid derivatives (CA: 100 μM; tauroursodeoxycholic acid TUDCA: 100 μM; deoxycholic acid DCA: 100 μM for hMSC, BHK-21, HT-1080, CHO-K1 and HEK-293, 10 μM for Freestyle 293F). SEAP levels in the culture supernatant were scored 24 h after addition of bile acids. Data presented are mean ± SD, n ⩾3. (F) Reversibilty of cholic acid-inducible SEAP expression. pPB2/pSP16-transgenic HEK-293 cells were cultivated in the presence (ON, for 6 h) or absence (OFF) of CA (100 μM). Every 24 h, the CA status of the culture was reversed and SEAP production was profiled for up to 72 h. Data presented are mean ± SD, n ⩾3. (This figure appears in colour on the web.)