Human TRPV1 Stable Cell Line-HEK293

Case Study 1

Transient receptor potential vanilloid 1 (TRPV1) is a noxious, thermosensitive, chemo-nociceptive cation channel expressed in primary sensory neurons of multimodal nociceptors. The present study was devoted to analyzing the role of lipid raft components in calcium influx induced by various TRPV1 agonists in sensory neurons and rTRPV1-transfected CHO cell lines. Depletion of cholesterol by methyl β-cyclodextrin (MCD, 1-10 mM) reduced the percentage of calcium uptake in cultured trigeminal neurons in response to capsaicin or resiniferatoxin (RTX, 3 nM). In contrast, in TRPV1-transfected cells, inhibition was observed only when capsaicin or N-oleoyl dopamine (OLDA, 10 µM) was applied, but not when gated with RTX, anandamide (AEA, 10 µM), or pH 5.5. Treatment of rTRPV1-expressing cells with sphingomyelinase inhibited capsaicin-induced 45Ca uptake, whereas RTX-induced responses remained unchanged. On the other hand, in trigeminal neurons, the effects of both compounds were inhibited by sphingomyelinase treatment. Inhibition of ganglioside biosynthesis by D-threo-1-phenyl-2-decanoylamino-3-morpholine-1-propanol (D-PDMP) or myriocyn also reduced calcium uptake induced by capsaicin or RTX in cultured trigeminal neurons and rTRPV1-expressing cells. The present study demonstrates that depletion of different components of lipid rafts inhibits gating of TRPV1 cation channels by various vanilloid and non-vanilloid agents. Evidence for supporting roles of cholesterol, sphingomyelin and gangliosides was obtained in both native and TRPV1-transfected cells.

Figure 1. Effect of combined administration of low concentrations of MCD and SMase on capsaicin- and RTX-induced Ca²⁺-accumulation. A: Results of radioactive ⁴⁵Ca-uptake experiments in TRPV1-expressing CHO cell line. Ca²⁺-influx is presented in % of control (untreated). B: Results of the same pretreatment on cultured trigeminal sensory neurons. Percentage of responding cells to capsaicin and RTX is presented (responsive cells vs. non-responsive cells). (Szőke É, et al. 2010)

Creative Biogene has successfully constructed TRPV1 over-expressing stable cell lines with HEK293, CHO, HeLa, 1321N1, U2OS, RH7777, etc. These TRPV1 stable cell lines have excellent in vitro assay sensitivity and reproducibility. Therefore, conducting experiments with our TRPV1 stable cell lines saves time and resources while providing more accurate results.

Case Study 2

CAE suppresses benzo[a]pyrene (B[a]P) effects on xenobiotic response element- (XRE-) mediated signaling in HaCaT cells.

Antagonizing Effects of Clematis apiifolia DC. Extract against Benzo[a]pyrene-Induced Damage to Human Keratinocytes

Paper 2019

Oxidative Medicine and Cellular Longevity | DOI

Background
Benzo[a]pyrene (B[a]P) is an environmental pollutant that damages human keratinocytes. This study investigated whether Clematis apiifolia DC. extract (CAE) protects HaCaT cells against B[a]P. CAE suppressed B[a]P-induced aryl hydrocarbon receptor (AhR) signaling, reducing XRE reporter activity, CYP1A1 expression, AhR nuclear translocation, Src phosphorylation, ROS production, and IL-8 secretion. CAE also activated Nrf2/ARE signaling, increasing Nrf2, NQO1, and HO-1 expression and Nrf2 nuclear translocation. Knockdown experiments showed AhR inhibition and Nrf2 activation occurred independently. CAE may protect skin from B[a]P-containing pollutants.
Key Findings
CAE inhibited B[a]P-induced AhR signaling in HaCaT keratinocytes by decreasing XRE reporter activity and CYP1A1 protein/mRNA expression. It also blocked B[a]P-induced nuclear translocation of AhR without affecting ARNT, and suppressed B[a]P-induced Src phosphorylation at Tyr416; the Src inhibitor dasatinib mimicked these effects. CAE reduced B[a]P-induced ROS generation and IL-8 production but did not directly scavenge H2O2-induced ROS. In parallel, CAE activated ARE signaling, increasing ARE reporter activity and expression of Nrf2, NQO1, and HO-1, and promoted Nrf2 nuclear translocation. Nrf2 knockdown attenuated CAE-induced ARE activation and antioxidant gene upregulation. AhR knockdown did not affect CAE-induced HO-1 expression, and Nrf2 knockdown did not alter CAE-mediated CYP1A1 inhibition, indicating independent mechanisms. These dual actions suggest CAE protects keratinocytes against B[a]P-induced oxidative and inflammatory damage.
Contribution
Creative Biogene supplied the HEK293-TRPV1-luciferase stable cell line listed in the cell culture and reagents section. This commercial cell line was available for luciferase-based assays, although the main mechanistic experiments used HaCaT keratinocytes. Creative Biogene's contribution was therefore limited to providing this biological reagent.