Since its first discovery as a bioactive phospholipid inducing potent platelet aggregation, platelet-activating factor (PAF) has been shown to be involved in a wide variety of inflammatory and allergic disease states. Many pharmacological studies in the 1980s and 1990s also showed that PAF induces endothelium-dependent vascular relaxation and contraction of various smooth muscles (SMs), including those in the airway, gastrointestinal organs, and uterus. However, since the late 1990s, there have been few reports on the SM contractions induced by PAF. The lower urinary tract (LUT), particularly the urinary bladder (UB) has attracted recent attention in SM pharmacology research because patients with LUT dysfunctions including overactive bladder are increasing as the population ages. In addition, recent clinical studies have implicated the substantial role of PAF in the inflammatory state in LUT because its production increases with smoking and with cancer. However, the effects of PAF on mechanical activities of LUT SMs including UBSM have not been investigated to date. Recently, we found that PAF very strongly increased mechanical activities of UBSM in guinea pigs and mice, and partly elucidated the possible mechanisms underlying these actions of PAF. In this review, we describe the effects of PAF on LUT SMs by introducing our recent findings obtained in isolated UBSMs and discuss the physiological and pathophysiological significance. We also introduce our data showing the effects of PAF on the SM mechanical activities of genital tissues (prostate and vas deferens).

The signal transducer and activator of transcription 3 (STAT3) protein is a key regulator of cell differentiation, proliferation, and survival in hematopoiesis, immune responses, and other biological systems. STAT3 transcriptional activity is strictly regulated through various mechanisms, such as phosphorylation and dephosphorylation. In this study, we attempted to identify novel phosphatases which regulate STAT3 activity in response to cytokine stimulations. To this end, leukemia inhibitory factor (LIF)/STAT3 dependent phosphatase induction was evaluated in the mouse hepatoma cell line Hepa1–6. After LIF stimulation, the expression of several atypical dual specific phosphatases (aDUSPs) was upregulated in Hepa1–6 cells. Among the LIF-induced aDUSPs, we focused on DUSP15 and clarified its functions in LIF/STAT3 signaling using RNA interference. DUSP15 knockdown decreased LIF-induced Socs3 mRNA expression and STAT3 translocation. Furthermore, loss of DUSP15 reduced the phosphorylation of STAT3 at Tyr705 and Janus family tyrosine kinase 1 (Jak1) at Tyr1034/1035 in response to LIF. The interaction between Jak1 and DUSP15 was observed in LIF-stimulated Hepa1–6 cells. We also demonstrated the suppression of granulocyte colony-stimulating factor (G-CSF)-mediated gp130/STAT3-dependent cell growth of Ba/F-G133 cells via DUSP15 knockdown. Therefore, DUSP15 functions as a positive feedback regulator in the Jak1/STAT3 signaling cascade.

Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic factor involved in multiple vital biological processes and a key mediator of gene transcription in response to cytokines, growth factors and aberrant activation of oncogenic signaling. STAT3 has two splicing isoforms, STAT3α and STAT3β, derived from alternative splicing of exon 23 within pre-mRNA. STAT3β differs from STAT3α by replacement of 55 amino-acid residues in the C-terminal transactivation domain with 7 specific amino acids. Thus, a shorter STAT3β was originally regarded as a dominant negative isoform of STAT3α. Recently accumulating evidence from independent studies have shown STAT3 splicing isoforms confer distinct and overlapping functions in many fundamental cellular regulatory steps such as cell differentiation, inflammatory responses, and cancer progression. However, relatively little is known about the mechanisms of STAT3 pre-mRNA splicing, and it remains undiscovered which chemical compounds or bioactive substances can induce the STAT3β expression. In this study, we generated a potent reporter for detection of alternative splicing of STAT3 pre-mRNA optimized for the screening of function-known chemical library, and successfully identified entinostat, a histone deacetylase inhibitor, as a novel inducer of STAT3β through modulating mRNA splicing. Our findings demonstrate that alternative splicing of STAT3 can be regulated by a compound, providing an important clue for understanding the regulation mechanisms of the expression balance of STAT3 isoforms in a chemical biology approach. Entinostat is likely to be a promising seed compound for elucidating how the higher ratio of STAT3β expression impacts on biological responses associated with Janus kinase (JAK)/STAT3 signaling pathway.

Siweixizangmaoru decoction (SXD) is widely used as an anti-rheumatoid arthritis (RA) in Tibet, however, the specific anti-inflammatory mechanism of SXD is still unclear. This research attempts to examine the efficacy and possible mechanisms of SXD in treating RA. The primary chemical components of SXD were identified using UHPLC-Q-Exactive Orbitrap MS. We established a lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammatory injury model to explore the anti-inflammatory mechanism of SXD and validated it through in vivo experiments. According to our research in vitro as well as in vivo, SXD exhibits anti-inflammatory qualities. SXD can suppress nitric oxide (NO) and pro-inflammatory factor production in RAW264.7 cells activated by LPS. The mechanism underlying this effect might be connected to the janus tyrosine kinase 2–signal transducer and activator of transcription 3 (JAK2/STAT3) and nuclear factor-κB (NF-κB) signaling pathways. In vivo, SXD alleviates joint swelling, decreases the generation of inflammatory factors in the serum, lowers oxidative stress, and improves joint damage. In short, SXD improves joint degeneration and lowers symptoms associated with RA by regulating inflammation via the suppression of NF-κB and JAK2/STAT3 signaling pathway activation.

The pathogenesis of stress-related disorders involves aberrant glucocorticoid secretion, and decreased pH and increased lactate in the brain are common phenotypes in several psychiatric disorders. Mice treated with glucocorticoids develop these phenotypes, but it is unclear how glucocorticoids affect brain pH. Therefore, we investigated the effect of corticosterone (CORT), the main glucocorticoid in rodents, on extracellular pH and lactate release in cultured astrocytes, which are the main glial cells that produce lactate in the brain. CORT treatment for one week decreased the extracellular pH and increased the extracellular lactate level via glucocorticoid receptors. CORT also increased the intracellular pyruvate level and upregulated pyruvate dehydrogenase kinase 4 (PDK4), while PDK4 overexpression increased extracellular lactate and decreased the extracellular pH. Furthermore, PDK4 inhibition suppressed the increase in extracellular lactate and the decrease in extracellular pH induced by CORT. These results suggest that increased lactate release via accumulation of intracellular pyruvate in astrocytes by chronic glucocorticoid exposure contributes to decreased brain pH.

Helicobacter pylori eradication is crucial in the treatment of peptic ulcers caused by H. pylori infection, a disease highly prevalent in Asia. We present a pooled analysis of two randomized, double-blind, double-dummy, phase 3 studies evaluating the efficacy and safety of vonoprazan-based bismuth-containing quadruple therapy for H. pylori eradication. Patients aged ≥18 years with endoscopically confirmed duodenal or gastric ulcers were randomized 1 : 1 to receive vonoprazan 20 mg or lansoprazole 30 mg once daily for up to 6 (duodenal ulcers) or 8 weeks (gastric ulcers). H. pylori–positive patients received vonoprazan- or lansoprazole-based bismuth-containing quadruple therapy for the first 2 weeks. H. pylori eradication was determined using the carbon-13 urea breath test at a follow-up visit 4 weeks post-treatment. The H. pylori eradication rate was 90.6% with vonoprazan vs. 85.2% with lansoprazole (difference: 5.4%; 95% confidence interval (CI): −0.1, 10.8). H. pylori eradication rates were 7.1% (95% CI: 1.4, 12.8) and 12.6% (95% CI: 3.9, 22.0) higher in patients aged <65 years and current smokers, respectively, with vonoprazan vs. lansoprazole. In the Chinese subpopulation, the H. pylori eradication rate was 92.0% with vonoprazan vs. 86.0% with lansoprazole (difference: 6.1%; 95% CI: 0.5, 11.7). Treatment-emergent adverse events occurred in 72.7 vs. 62.6% of H. pylori–positive patients at baseline in the vonoprazan vs. lansoprazole arm. H. pylori eradication with vonoprazan-based quadruple therapy was noninferior to lansoprazole-based quadruple therapy and exceeded 90%, a clinically relevant threshold for determining the efficacy of H. pylori eradication regimens (ClinicalTrials.gov identifier: NCT03050359; NCT03050307).

The efficacy of mesenchymal stem cell (MSC) transplantation has been reported for various diseases. We previously developed a drug delivery system targeting mitochondria (MITO-Porter) by using a microfluidic device to encapsulate Coenzyme Q₁₀ (CoQ₁₀) on a large scale. The current study aimed to confirm if treatment with CoQ₁₀ encapsulated by MITO-Porter enhanced mitochondrial functions in MSCs, with the potential to improve MSC transplantation therapy. We used highly purified human bone marrow-derived MSCs, described as rapidly expanding clones (RECs), and attempted to control and increase the amount of CoQ₁₀ encapsulated in the MITO-Porter using microfluidic device system. We treated these RECs with CoQ₁₀ encapsulated MITO-Porter, and evaluated its cellular uptake, co-localization with mitochondria, changes in mitochondrial respiratory capacity, and cellular toxicity. There was no significant change in mitochondrial respiratory capacity following treatment with the previous CoQ₁₀ encapsulated MITO-Porter; however, mitochondrial respiratory capacity in RECs was significantly increased by treatment with CoQ₁₀-rich MITO-Porter. Utilization of a microfluidic device enabled the amount of CoQ₁₀ encapsulated in MITO-Porter to be controlled, and treatment with CoQ₁₀-rich MITO-Porter successfully activated mitochondrial functions in MSCs. The MITO-Porter system thus provides a promising tool to improve MSC cell transplantation therapy.

CYP2D6 variants contain various single nucleotide polymorphisms as well as differing levels of metabolic activity. Among these, one of the less active variants CYP2D6*10 (100C > T) is the most prevalent mutation in East Asians, including Japanese. This mutation leads to an amino acid substitution from proline to serine, which reduces the stability of CYP2D6 and consequently decreases its metabolic activity. In this study, we used a genome editing technology called the Precise Integration into Target Chromosome (PITCh) system to stably express six drug-metabolizing enzymes (CYP3A4, POR, uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), CYP1A2, CYP2C19, CYP2C9, and CYP2D6*10) in HepG2 (CYP2D6*10 KI-HepG2) cells to examine the effect of CYP2D6*10 on drug metabolism prediction. The protein expression levels of CYP2D6 in CYP2D6*10 KI-HepG2 cells were reduced relative to those in the CYP3A4-POR-UGT1A1-CYP1A2-CYP2C19-CYP2C9-CYP2D6 knock-in-HepG2 (CYPs-UGT1A1 KI-HepG2) cells. Consistent with the CYP2D6 protein expression results, CYP2D6 metabolic activity in CYP2D6*10 KI-HepG2 cells was reduced relative to CYPs-UGT1A1 KI-HepG2 cells. We successfully generated CYP2D6*10 KI-HepG2 cells with highly expressed, functional CYP2D6*10, as well as CYP1A2, 2C9, 2C19 and 3A4. CYP2D6*10 KI-HepG2 cells could be an invaluable model for hepatic metabolism and hepatotoxicity studies in East Asians, including Japanese.

Bisdemethoxycurcumin (BDMC) is one of major forms of curcuminoids found in the rhizomes of turmeric. Docetaxel (DTX) is the standard of care for men diagnosed with androgen-independent prostate cancers. Here we report for the first time that BDMC could reinforce the effect of DTX against prostate cancer in vitro and in vivo. In vitro study, PC3 and LNCaP cells were cultured and treated with BDMC and DTX alone or in combination. The effects on cell viability were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was assessed by annexin V/propidium iodide (PI) staining, while cell cycle was assessed by PI staining. Bax, Bcl-2, caspase, poly(ADP-ribose)polymerase (PARP), cyclin B1 and CDK1 expression were assayed by Western blot. We found that a combination treatment of BDMC (10 µM) with DTX (10 nM) was more effective in the inhibition of PC3 and LNCaP cell growth and induction of apoptosis as well as G2/M arrest, which is accompanied with the significant inhibition of Bcl-2, cyclin B1, CDK1 expression and significant increase of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, than those by treatment of BDMC or DTX alone. Moreover, in vivo evaluation further demonstrated the superior anticancer efficacy of BDMC and DTX compared to DTX alone in a murine prostate cancer model. These results suggest that BDMC can be an attractive therapeutic candidate in enhancing the efficacy of DTX in prostate cancer treatment.

Research on sex differences has increased across various fields, including cancer and its treatment domains. Reports have indicated sex differences in cancer incidence, survival rates, and the efficacy of anticancer drugs. However, such reports are limited, and in-depth assessments of the underlying mechanisms are still in progress. Although various chemotherapeutic regimens are applicable for breast cancer treatment, reports have surfaced regarding weight gain in female patients undergoing fluorouracil, epirubicin, cyclophosphamide (FEC) or cyclophosphamide, methotrexate, fluorouracil (CMF) therapy. We hypothesized the potential of 5-fluorouracil (5-FU) in weight gain and sex-related differences. To address this, we conducted experiments in mice to confirm weight gain and sex differences following 5-FU administration, and elucidate the underlying mechanisms. Our findings revealed weight gain and increased food intake in female mice following 5-FU administration. Additionally, female mice receiving 5-FU exhibited increased norepinephrine and α1- and α2-adrenergic receptor expression, reduced estradiol levels, and increased ghrelin levels. These results indicate 5-FU administration-induced sex differences in weight gain and implicate increased food intake because of increased norepinephrine and α1- and α2-adrenergic receptor expression, reduced estradiol levels, and a subsequent increase in ghrelin levels, which contribute to weight gain in female patients undergoing CMF therapy.

Assay systems for evaluating compound protein-binding affinities are essential for developing agonists and/or antagonists. Targeting individual members of a protein family can be extremely important and for this reason it is critical to have methods for evaluating selectivity. We have previously reported a fluorescence recovery assay that employs a fluorescein-labelled probe to determine IC₅₀ values of ATP-competitive type 1 inhibitors of polo-like kinase 1 (Plk1). This probe is based on the potent Plk1 inhibitor BI2536 [fluorescein isothiocyanate (FITC)-polyethylene glycol (PEG)-lysine (Lys) (BI2536) 1]. Herein, we extend this approach to the highly homologous Plk2 and Plk3 members of this kinase family. Our results suggest that this assay system is suitable for evaluating binding affinities against Plk2 and Plk3 as well as Plk1. The new methodology represents the first example of evaluating N-terminal catalytic kinase domain (KD) affinities of Plk2 and Plk3. It represents a simple and cost-effective alternative to traditional kinase assays to explore the KD-binding compounds against Plk2 and Plk3 as well as Plk1.

Dab1 is an intracellular adaptor protein essential for brain formation during development. Tyrosine phosphorylation in Dab1 plays important roles in neuronal migration, dendrite development, and synapse formation by affecting several downstream pathways. Reelin is the best-known extracellular protein that induces Dab1 phosphorylation. However, whether other upstream molecule(s) contribute to Dab1 phosphorylation remains largely unknown. Here, we found that EphA4, a member of the Eph family of receptor-type tyrosine kinases, induced Dab1 phosphorylation when co-expressed in cultured cells. Tyrosine residues phosphorylated by EphA4 were the same as those phosphorylated by Reelin in neurons. The autophosphorylation of EphA4 was necessary for Dab1 phosphorylation. We also found that EphA4-induced Dab1 phosphorylation was mediated by the activation of the Src family tyrosine kinases. Interestingly, Dab1 phosphorylation was not observed when EphA4 was activated by ephrin-A5 in cultured cortical neurons, suggesting that Dab1 is localized in a different compartment in them. EphA4-induced Dab1 phosphorylation may occur under limited and/or pathological conditions in the brain.

The Accelerated Approval (AA) Program of the United States (US) Food and Drug Administration (FDA) was established to facilitate and expedite access to new drugs for serious or life-threatening conditions. Although many drugs have granted AAs in the US, the number of approvals under the conditional approval system in Japan remains limited. This study aimed to examine whether confirmatory trials after the US AA are conducted in accordance with the design of postmarketing requirements and assess the timing of regular approval (RA) in Japan for drugs that have been granted US AA. Utilizing FDA databases and Japanese regulatory data from 1992 to 2023, we analyzed indications, postmarketing requirements, and clinical trial designs. Our findings indicate that the AA program in the US is well-managed as most AAs were converted to RAs based on confirmatory study data that met the designations. From the Japanese perspective, our findings show that the over half of Japanese RAs can be obtained without waiting for confirmatory trial results. By granting RA, instead of conditional approval, based on exploratory trial data in the US AA, the opportunity to evaluate postmarketing confirmatory trial results might be lost in Japan. Therefore, further improvements are needed to actively utilize the conditional approval system, which could allow for the rapid introduction of innovative drugs and also the verification of their efficacy and safety at an appropriate time.

We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K⁺ concentration in rat caudal arterial smooth muscle involves a Ca²⁺-induced Ca²⁺ sensitization mechanism whereby Ca²⁺ entry through voltage-gated Ca²⁺ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) induced by a Ca²⁺ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca²⁺. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC₂₀) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca²⁺]_i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca²⁺]_i elevation.

18-β-Glycyrrhetinic acid, a major component of licorice, stimulated the proliferation of both dermal papilla cells and outer root sheath cells isolated from human hair follicles. Thus, suggesting that this compound promotes hair growth. Furthermore, this compound inhibited the activity of testosterone 5α-reductase, an enzyme responsible for converting androgen to dihydroandrogen, with an IC₅₀ of 137.1 µM. 18-β-Glycyrrhetinic acid also suppressed the expression of transforming growth factor-β1 (TGF-β1), which shifts the hair cycle from the anagen phase to the telogen phase. It suggested that this compound may prolong the anagen phase. Based on these findings, this compound could be a potentially effective treatment for androgenetic alopecia.

DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.

Transcriptional activation, based on Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) and known as CRISPR activation (CRISPRa), is a specific and safe tool to upregulate endogenous genes. Therefore, CRISPRa is valuable not only for analysis of molecular mechanisms of cellular events, but also for treatment of various diseases. Regulating autophagy has been proposed to enhance effects of some therapies. In this study, we upregulated genes for phosphoinositide phosphatases, SACM1L, PIP4P1, and PIP4P2, using CRISPRa, and their effects on autophagy were examined. Our results suggested that TMEM55A/PIP4P2, a phosphatidylinositol-4,5-bisphosphate 4-phosphatase, positively regulates basal autophagy in 293A cells. Furthermore, it was also suggested that SAC1, a phosphatidylinositol 4-phosphatase, negatively regulates basal autophagic degradation.

The increasing number of patients with depressive disorder is a serious socioeconomic problem worldwide. Although several therapeutic agents have been developed and used clinically, their effectiveness is insufficient and thus discovery of novel therapeutic targets is desired. Here, focusing on dysregulation of neuronal purinergic signaling in depressive-like behavior, we examined the expression profiles of ATP channels and ectonucleotidases in astrocytes of cerebral cortex and hippocampus of chronic social defeat stress (CSDS)-susceptible BALB/c mice. Mice were exposed to 10-d CSDS, and their astrocytes were obtained using a commercially available kit based on magnetic activated cell sorting technology. In astrocytes derived from cerebral cortex of CSDS-susceptible mice, the expression levels of mRNAs for connexin 43, P2X7 receptors and maxi anion channels were increased, those for connexin 43 and P2X7 receptors being inversely correlated with mouse sociability, and the expression of mRNAs for ecto-nucleoside triphosphate diphosphohydrase 2 and ecto-5′nucleotidase was decreased and increased, respectively. On the other hand, the alteration profiles of ATP channels and ectonucleotidases in hippocampal astrocytes of CSDS-susceptible mice were different from in the case of cortical astrocytes, and there was no significant correlation between expression levels of their mRNAs and mouse sociability. These findings imply that increased expression of ATP channels in cerebral cortex might be involved in the development of reduced sociability in CSDS-subjected BALB/c mice. Together with recent findings, it is suggested that ATP channels expressed by cortical astrocytes might be potential therapeutic targets for depressive disorder.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

Unknown interactions between drugs remain the limiting factor for clinical application of drugs, and the induction and inhibition of drug-metabolizing CYP enzymes are considered the key to examining the drug–drug interaction (DDI). In this study, using human HepaRG cells as an in vitro model system, we analyzed the potential DDI based on the expression levels of CYP3A4 and CYP1A2. Rifampicin and omeprazole, the potent inducers for CYP3A4 and CYP1A2, respectively, induce expression of the corresponding CYP enzymes at both the mRNA and protein levels. We noticed that, in addition to inducing CYP1A2, omeprazole induced CYP3A4 mRNA expression in HepaRG cells. However, unexpectedly, CYP3A4 protein expression levels were not increased after omeprazole treatment. Concurrent administration of rifampicin and omeprazole showed an inhibitory effect of omeprazole on the CYP3A4 protein expression induced by rifampicin, while its mRNA induction remained intact. Cycloheximide chase assay revealed increased CYP3A4 protein degradation in the cells exposed to omeprazole. The data presented here suggest the potential importance of broadening the current DDI examination beyond conventional transcriptional induction and enzyme-activity inhibition tests to include post-translational regulation analysis of CYP enzyme expression.