Flavonoids activate pregnane × receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells

<p>Abstract</p> <p>Background</p> <p>The expression of the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is regulated by the pregnane × receptor (PXR), which is modulated by numerous signaling pathways, including the cyclin-dependent kinase (Cdk) pathway. Flavonoids, commonly consumed by humans as dietary constituents, have been shown to modulate various signaling pathways (e.g., inhibiting Cdks). Flavonoids have also been shown to induce <it>CYPs </it>expression, but the underlying mechanism of action is unknown. Here, we report the mechanism responsible for flavonoid-mediated PXR activation and <it>CYP </it>expression.</p> <p>Results</p> <p>In a cell-based screen designed to identify compounds that activate PXR-mediated <it>CYP3A4 </it>gene expression in HepG2 human carcinoma cells, we identified several flavonoids, such as luteolin and apigenin, as PXR activators. The flavonoids did not directly bind to PXR, suggesting that an alternative mechanism may be responsible for flavonoid-mediated PXR activation. Consistent with the Cdk5-inhibitory effect of flavonoids, Cdk5 and p35 (a non-cyclin regulatory subunit required to activate Cdk5) were expressed in HepG2. The activation of Cdk5 attenuated PXR-mediated <it>CYP3A4 </it>expression whereas its downregulation enhanced it. The Cdk5-mediated downregulation of <it>CYP3A4 </it>promoter activity was restored by flavonoids, suggesting that flavonoids activate PXR by inactivating Cdk5. <it>In vitro </it>kinase assays showed that Cdk5 directly phosphorylates PXR. The Cdk kinase profiling assay showed that apigenin inhibits multiple Cdks, suggesting that several Cdks may be involved in activation of PXR by flavonoids.</p> <p>Conclusions</p> <p>Our results for the first time link the stimulatory effect of flavonoids on <it>CYP </it>expression to their inhibitory effect on Cdks, through a PXR-mediated mechanism. These results may have important implications on the pharmacokinetics of drugs co-administered with herbal remedy and herbal-drug interactions.</p

Modeling autosomal dominant optic atrophy using induced pluripotent stem cells and identifying potential therapeutic targets

OPA1 +/− -iPSCs (OL) are unfavorable to differentiate into neural rosette. Culture conditions were the same as those in Fig. 3. Panel A shows day 5 EBs, which were derived from control and OL-iPSCs, respectively. Morphologically, OL-EBs look similar to control EBs. Panel B shows neuron rosettes (NRs) after 5 days of EB attachment. Fewer and smaller neuron rosettes were derived from OL-EBs, compared with neuron rosettes from control EBs. Figure S2 OPA1 +/− -iPSCs (OL) failed to differentiate into RGCs with culture medium supplemented with 10 % FBS and DAPT. Differentiation method in Fig. 4 was followed. Putative RGCs were fixed on day 24 before fixation, followed by IF staining. Upper panel shows staining of TUJ1 (green) and BRN3a (red), while lower panel displays staining of TUJ1 (green) and ISLET1 (red). The scale bars equal 50 μM. Figure S3 IF analysis of RGCs derived from the control and VO-iPSCs on day 38. Neurospheres were plated onto PLO/L-coated plates and cultured in hESC medium containing 10 μM DAPT and 10 % FBS for 21 days before fixation with 4 % PFA on day 38. Cells were stained with TUJ1 (green), BRN3a (red) and ISLET-1 (red) antibodies. The scale bars equal 20 μM. Figure S4 NIM promoted OPA1 +/− -RGC (OL) generation detected by IF. Putative OL-RGCs derived from OL-iPSCs were cultured with hESC medium containing 10 μM DAPT, 10 % FBS, and 10 % neural induction medium (NIM) for 14 days before fixation on day 31. Upper panel shows TUJ1 (green) and BRN3a (red) staining. Lower panel shows staining of TUJ1 (green) and ISLET1 (red). The scale bars equal 50 μM. Figure S5 Quantification of RGC differentiation efficiency. The culture medium used for RGC differentiation contained 10 % neural induction medium. Samples were normalized to the control BRN3a/DAPI staining or the control ISLET-1/DAPI staining. The number of BRN3a or ISLET-1 signals versus the number of DAPI signals was calculated. A p-value of 0.084 was obtained for the BRN3a signal comparison between the control and VO, and a p-value of 0.076 was obtained for the ISLET-1 signal comparison between the control and VO. Student’s t-test was used to analyze differences between two groups. Figure S6 Noggin rescued OPA1 +/− -iPSC (OL) differentiation into RGCs confirmed by IF. Mature EBs were cultured with hES medium containing 10 % FBS and 100 ng/mL Noggin to obtain neuron rosettes, followed by culturing putative RGCs with hES medium containing 10 % FBS, DAPT and 100 ng/mL Noggin. Cells were fixed with 4 % PFA on day 31. Upper panel shows IF staining of TUJ-1 (green) and BRN3a (red), whereas lower panel shows staining of TUJ-1 (green) and ISLET1 (red). The scale bars equal 50 μM in both panels. Figure S7 Addition of 17β-estradiol in RGC differentiation medium promoted generation of OPA1 +/− -RGCs (OL). Putative RGCs were incubated with 100 nM 17β-estradiol in the cell culture medium during all of the differentiation stages, followed by fixation on day 31. Upper panel shows IF staining against TUJ1 (green) and BRN3a (red). Lower panel shows TUJ1 (green) and ISLET-1 (red) staining. The scale bars equal 50 μM. (PPTX 12904 kb

A tea catechin, epigallocatechin-3-gallate, is a unique modulator of the farnesoid X receptor

Farnesoid X receptor (FXR) is a ligand-activated nuclear receptor and serves as a key regulator to maintain health of the liver and intestine. Bile acids are endogenous ligands of FXR, and there are increasing efforts to identify FXR modulators to serve as biological probes and/or pharmaceutical agents. Natural FXR ligands isolated from plants may serve as models to synthesize novel FXR modulators. In this study, we demonstrated that epigallocatechin-3-gallate (EGCG), a major tea catechin, specifically and dose-dependently activates FXR. In addition, EGCG induced FXR target gene expression in vitro. Surprisingly, in a co-activator (SRC2) recruitment assay, we found that EGCG does not recruit SRC2 to FXR, but it dose-dependently inhibits recruitment of SRC2 to FXR (IC50, 1 μM) by GW6064, which is a potent FXR synthetic ligand. In addition, EGCG suppressed FXR target gene expression induced by either GW4064 or chenodeoxycholic acid in vitro. Furthermore, wild-type and FXR knockout mice treated with an acute dose of EGCG had induced mRNA expression in a subset of FXR target genes in the intestine but not in the liver. In conclusion, EGCG is a unique modulator of FXR in the intestine and may serve as an important model for future development of FXR modulators

Metabolic activation of CaMKII by coenzyme A

Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca(2+) to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability

Heterozygous Mutation of Drosophila Opa1 Causes the Development of Multiple Organ Abnormalities in an Age-Dependent and Organ-Specific Manner

Optic Atrophy 1 (OPA1) is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal dominant optic atrophy (DOA). The molecular mechanisms by which link OPA1 mutations and DOA are not fully understood. Recently, we created a Drosophila model to study the pathogenesis of optic atrophy. Heterozygous mutation of Drosophila OPA1 (dOpa1) by P-element insertion results in no obvious morphological abnormalities, whereas homozygous mutation is embryonic lethal. In eye-specific somatic clones, homozygous mutation of dOpa1 causes rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult Drosophila. In humans, heterozygous mutations in OPA1 have been associated with mitochondrial dysfunction, which is predicted to affect multiple organs. In this study, we demonstrated that heterozygous dOpa1 mutation perturbs the visual function and an ERG profile of the Drosophila compound eye. We independently showed that antioxidants delayed the onset of mutant phenotypes in ERG and improved larval vision function in phototaxis assay. Furthermore, heterozygous dOpa1 mutation also caused decreased heart rate, increased heart arrhythmia, and poor tolerance to stress induced by electrical pacing. However, antioxidants had no effects on the dysfunctional heart of heterozygous dOpa1 mutants. Under stress, heterozygous dOpa1 mutations caused reduced escape response, suggesting abnormal function of the skeletal muscles. Our results suggest that heterozygous mutation of dOpa1 shows organ-specific pathogenesis and is associated with multiple organ abnormalities in an age-dependent and organ-specific manner