Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

Metabolism Pathways of Major Therapeutics for Treating Monkeypox Mono- and Co-infection with Human Immunodeficient Virus or SARS-CoV-2.

Monkeypox is a zoonotic viral disease and remains endemic in tropical regions of Central and West Africa. Since May of 2022, cases of monkeypox have soared and spread worldwide. Confirmed cases have shown no travel history to the endemic regions as seen in the past. The World Health Organization declared monkeypox a global public health emergency in July 2022, and the United States government followed suit one month later. The current outbreak, in contrast to traditional epidemics, has high coinfection rates, particularly with HIV (human immunodeficiency virus), and to a lesser extent with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the pathogen of COVID-19. No drugs have been approved specifically for monkeypox. However, there are therapeutic agents authorized to treat monkeypox under the Investigational New Drug protocol, including brincidofovir, cidofovir, and tecovirimat. In contrast to limited options for monkeypox treatment, there are available drugs specifically for HIV or SARS-CoV-2 infection. Interestingly, these HIV and COVID-19 medicines share metabolism pathways with those authorized to treat monkeypox, particularly of hydrolysis, phosphorylation, and active membrane transport. This review discusses how these pathways shared by these medicines should be considered to gain therapeutic synergy and maximize safety for treating monkeypox coinfections.

Viral target and metabolism-based rationale for combined use of recently authorized small molecule COVID-19 medicines: Molnupiravir, nirmatrelvir, and remdesivir.

The COVID-19 pandemic remains a major health concern worldwide, and SARS-CoV-2 is continuously evolving. There is an urgent need to identify new antiviral drugs and develop novel therapeutic strategies. Combined use of newly authorized COVID-19 medicines including molnupiravir, nirmatrelvir, and remdesivir has been actively pursued. Mechanistically, nirmatrelvir inhibits SARS-CoV-2 replication by targeting the viral main protease (Mpro ), a critical enzyme in the processing of the immediately translated coronavirus polyproteins for viral replication. Molnupiravir and remdesivir, on the other hand, inhibit SARS-CoV-2 replication by targeting RNA-dependent RNA-polymerase (RdRp), which is directly responsible for genome replication and production of subgenomic RNAs. Molnupiravir targets RdRp and induces severe viral RNA mutations (genome), commonly referred to as error catastrophe. Remdesivir, in contrast, targets RdRp and causes chain termination and arrests RNA synthesis of the viral genome. In addition, all three medicines undergo extensive metabolism with strong therapeutic significance. Molnupiravir is hydrolytically activated by carboxylesterase-2 (CES2), nirmatrelvir is inactivated by cytochrome P450-based oxidation (e.g., CYP3A4), and remdesivir is hydrolytically activated by CES1 but covalently inhibits CES2. Additionally, remdesivir and nirmatrelvir are oxidized by the same CYP enzymes. The distinct mechanisms of action provide strong rationale for their combined use. On the other hand, these drugs undergo extensive metabolism that determines their therapeutic potential. This review discusses how metabolism pathways and enzymes involved should be carefully considered during their combined use for therapeutic synergy.

Differentiated embryonic chondrocyte expressed gene-1 is a central signaling component in the development of collagen-induced rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases and affects almost 1% of the population. Differentiated embryo-chondrocyte expressed gene-1 (DEC1) has been associated with both osteogenesis and osteoclastogenesis. RA condition is marked by inflammatory hyperplasia, and DEC1 is known to support inflammatory reactions and implicated in antiapoptosis and cell invasion. Here, our goal was to test the hypothesis that DEC1 enhances RA development induced by collagen-induced arthritis (CIA), a well-recognized protocol for developing RA animal models. DEC1+/+ and DEC1-/- mice were subjected to CIA protocol, and the development of RA condition was monitored. We found that CIA robustly induced RA phenotypes (e.g., synovial hyperplasia) and greatly increased the expression of proinflammatory cytokines such as TNF-α. However, these changes were detected in DEC1+/+ but not DEC1-/- mice. Interestingly, these very cytokines strongly induced DEC1, and such a dual role of DEC1, as an inducer for and being induced by proinflammatory cytokines, constitutes a DEC1-amplifying circuit for inflammation. Knockdown of DEC1 in human MH7A cells strongly decreased cell migration and invasion as well as the expression of genes related to RA phenotypes. The combination of DEC1-directed migration and invasion in vitro with synovial hyperplasia in vivo mechanistically establishes cellular bases on how DEC1 is involved in the development of RA phenotypes. In addition to inflammatory signaling, DEC1 functionally interacted with PI3KCA(p110α)/Akt/GSK3ß, Wnt/ß-catenin, and NFATc1. Such engagement in multiple signaling pathways suggests that DEC1 plays coordinated and integral roles in developing RA, one of the most common autoimmune diseases.

Oral Drug Delivery via Intestinal Lymphatic Transport Utilizing Lipid-Based Lyotropic Liquid Crystals.

Lyotropic liquid crystals (LLCs) are liquids that have crystalline structures. LLCs as drug delivery systems that can deliver hydrophobic, hydrophilic, and amphiphilic agents. Due to their unique phases and structures, LLCs can protect both small molecules and biologics from the gastrointestinal tract's harsh environment, thus making LLCs attractive as carriers for oral drug delivery. In this review, we discuss the advantages of LLCs and LLCs as oral formulations targeting intestinal lymphatic transport. In oral LLC formulations, the relationship between the micelle compositions and the resulting LLC structures as well as intestinal transport and absorption were determined. In addition, we further demonstrated approaches for the enhancement of intestinal lymphatic transport: (1) lipid-based LLCs promoting chylomicron secretion and (2) the design of LLC nanoparticles with M cell-triggered ligands for targeting the M cell pathway. In this review, we introduce LLC drug delivery systems and their characteristics. Our review focuses on recent approaches using oral LLC drug delivery strategies targeting the intestinal lymphatic system to enhance drug bioavailability.

Covalent CES2 Inhibitors Protect against Reduced Formation of Intestinal Organoids by the Anticancer Drug Irinotecan.

BACKGROUND: Irinotecan is widely used to treat various types of solid and metastatic cancer. It is an ester prodrug and its hydrolytic metabolite (SN-38) exerts potent anticancer activity. Irinotecan is hydrolyzed primarily by carboxylesterase-2 (CES2), a hydrolase abundantly present in the intestine such as the duodenum. We have identified several potent and covalent CES2 inhibi¬tors such as remdesivir and sofosbuvir. Remdesivir is the first small molecule drug approved for COVID-19, whereas sofosbuvir is a paradigm-shift medicine for hepatitis C viral infection. Irinotecan is generally well-tolerated but associated with severe/life-threatening diarrhea due to intestinal accu¬¬mula¬tion of SN-38. OBJECTIVE: This study was to test the hypothesis that remdesivir and sofosbuvir protect against irinotecan-induced epithelial injury associated with gastrointestinal toxicity. METHODS: To test this hypothesis, formation of organoids derived from mouse duodenal crypts, a robust cellular model for intestinal regeneration, was induced in the presence or absence of irinotecan +/- pretreatment with a CES2 drug inhibitor. RESULTS: Irinotecan profoundly inhibited the formation of intestinal organoids and the magnitude of the inhibition was greater with female crypts than their male counterparts. Consistently, crypts from female mice had significantly higher hydrolytic activity toward irinotecan. Critically, remdesivir and sofosbuvir both reduced irinotecan hydrolysis and reversed irinotecan-reduced formation of organoids. Human duodenal samples robustly hydrolyzed irinotecan, stable CES2 transfection induced cytotoxicity and the cytotoxicity was reduced by CES2 drug inhibitor. CONCLUSION: These findings establish a therapeutic rationale to reduce irinotecan-gastrointestinal injury and serve as a cellular foundation to develop oral formulations of irinotecan with high safety.

Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development.

In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and ß may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients' tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient's tumor tissues are used to develop a spheroid culture for drug screening.

Effect of alcohol exposure on the efficacy and safety of tenofovir alafenamide fumarate, a major medicine against human immunodeficiency virus.

Human immunodeficiency virus (HIV) continues to be a major health concern. AIDS-related deaths (acquired immunodeficiency syndrome) have decreased recently, but chronic liver disease is now a major cause of mortality among HIV patients. Widespread alcohol use is recognized to be a major contributing factor. Tenofovir alafenamide fumarate (TAF), one of the most used HIV drugs, requires hydrolysis followed by phosphorylation to produce tenofovir diphosphate, the ultimate anti-HIV metabolite. Carboxylesterase-1 (CES1), established to hydrolyze TAF, is known to catalyze transesterification in the presence of ethanol. The aim of the study was to test the hypothesis that metabolism-based interactions between TAF and ethanol negatively impact both efficacy and safety of TAF. To test this hypothesis, the metabolism of TAF was determined in human primary hepatocytes and with a large number of human liver samples (S9 fractions) in the presence or absence of ethanol. The metabolism was monitored by LC-MS/MS (liquid chromatography with tandem mass spectrometry) and the level of CES1 or CES2 was determined by Western blotting. Consistent with the hypothesis, TAF underwent transesterification in the presence of ethanol accompanied by decreased hydrolysis. The formation of tenofovir diphosphate (the therapeutically active metabolite) was significantly decreased. In addition, TAF but not its hydrolytic metabolite, was found to increase intracellular lipid retention, and the increase was enhanced by ethanol. These findings conclude that alcohol consumption, beyond commonly accepted poor adherence to HIV medications, directly impacts the efficacy and safety of TAF.

The COVID-19 Oral Drug Molnupiravir Is a CES2 Substrate: Potential Drug-Drug Interactions and Impact of CES2 Genetic Polymorphism In Vitro.

Molnupiravir is one of the two coronavirus disease 2019 (COVID-19) oral drugs that were recently granted the emergency use authorization by the Food and Drug Administration (FDA). Molnupiravir is an ester and requires hydrolysis to exert antiviral activity. Carboxylesterases constitute a class of hydrolases with high catalytic efficiency. Humans express two major carboxylesterases (CES1 and CES2) that differ in substrate specificity. Based on the structural characteristics of molnupiravir, this study was performed to test the hypothesis that molnupiravir is preferably hydrolyzed by CES2. Several complementary approaches were used to test this hypothesis. As many as 24 individual human liver samples were tested and the hydrolysis of molnupiravir was significantly correlated with the level of CES2 but not CES1. Microsomes from the intestine, kidney, and liver, but not lung, all rapidly hydrolyzed molnupiravir and the magnitude of hydrolysis was related closely to the level of CES2 expression among these organs. Importantly, recombinant CES2 but not CES1 hydrolyzed molnupiravir, collectively establishing that molnupiravir is a CES2-selective substrate. In addition, several CES2 polymorphic variants (e.g., R180H) differed from the wild-type CES2 in the hydrolysis of molnupiravir. Molecular docking revealed that wild-type CES2 and its variant R180H used different sets of amino acids to interact with molnupiravir. Furthermore, molnupiravir hydrolysis was significantly inhibited by remdesivir, the first COVID-19 drug granted the full approval by the FDA. The results presented raise the possibility that CES2 expression and genetic variation may impact therapeutic efficacy in clinical situations and warrants further investigation. SIGNIFICANCE STATEMENT: COVID-19 remains a global health crisis, and molnupiravir is one of the two recently approved oral COVID-19 therapeutics. In this study, we have shown that molnupiravir is hydrolytically activated by CES2, a major hydrolase whose activity is impacted by genetic polymorphic variants, disease mediators, and many potentially coadministered medicines. These results presented raise the possibility that CES2 expression and genetic variation may impact therapeutic efficacy in clinical situations and warrants further investigation.

Differentiated embryonic chondrocyte expressed gene-1 (DEC1) enhances the development of colorectal cancer with an involvement of the STAT3 signaling.

Colorectal cancer (CRC) is the second deadly and the third most common malignancy worldwide. It has been projected that annual new cases of CRC will increase by 63% in 2040, constituting an even greater health challenge for decades to come. This study has linked DEC1 (differentiated embryonic chondrocyte expressed gene 1) to the pathogenesis of CRC. Based on the analysis of patient samples and database data, DEC1 is expressed much higher in CRC than the adjacent normal tissues. CRC patients with higher DEC1 expression have a shorter survival time. The carcinogenesis protocol with azoxymethane/dextran sulfate induces a higher number of tumors with larger sizes in DEC1+/+ than DEC1-/- mice. Overexpression of DEC1 increases the expression of proliferation- and antiapoptosis-related genes, but decreases the level of proapoptotic genes. Mechanistically, this study has shown that DEC1 is functionally looped to the IL-6/STAT3 signaling pathway (interleukin-6/signal transducer and activator of transcription 3). IL-6 induces DEC1, and DEC1 enhances the phosphorylation of STAT3, resulting in increased pSTAT3/STAT3 ratio. DEC1 and STAT3 are present in reciprocal immunocomplexes, pointing to physical interactions (presumably with pSTAT3). These findings establish that DEC1 is a CRC enhancer. The enhancement is achieved largely through the IL-6/STAT3 pathway. The potential of the physical interaction between DEC1 and STAT3 will likely serve as a foundation to develop intervention strategies for CRC prevention and therapy.

The nucleoside antiviral prodrug remdesivir in treating COVID-19 and beyond with interspecies significance.

Infectious pandemics result in hundreds and millions of deaths, notable examples of the Spanish Flu, the Black Death and smallpox. The current pandemic, caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), is unprecedented even in the historical term of pandemics. The unprecedentedness is featured by multiple surges, rapid identification of therapeutic options and accelerated development of vaccines. Remdesivir, originally developed for Ebola viral disease, is the first treatment of COVID-19 (Coronavirus disease 2019) approved by the United States Food and Drug Administration. As demonstrated by in vitro and preclinical studies, this therapeutic agent is highly potent with a broad spectrum activity against viruses from as many as seven families even cross species. However, randomized controlled trials have failed to confirm the efficacy and safety. Remdesivir improves some clinical signs but not critical parameters such as mortality. This antiviral agent is an ester/phosphorylation prodrug and excessive hydrolysis which increases cellular toxicity. Remdesivir is given intravenously, leading to concentration spikes and likely increasing the potential of hydrolysis-based toxicity. This review has proposed a conceptual framework for improving its efficacy and minimizing toxicity not only for the COVID-19 pandemic but also for future ones caused by remdesivir-sensitive viruses.

Pig Liver Esterases Hydrolyze Endocannabinoids and Promote Inflammatory Response.

Endocannabinoids are endogenous ligands of cannabinoid receptors and activation of these receptors has strong physiological and pathological significance. Structurally, endocannabinoids are esters (e.g., 2-arachidonoylglycerol, 2-AG) or amides (e.g., N-arachidonoylethanolamine, AEA). Hydrolysis of these compounds yields arachidonic acid (AA), a major precursor of proinflammatory mediators such as prostaglandin E2. Carboxylesterases are known to hydrolyze esters and amides with high efficiency. CES1, a human carboxylesterase, has been shown to hydrolyze 2-AG, and shares a high sequence identity with pig carboxylesterases: PLE1 and PLE6 (pig liver esterase). The present study was designed to test the hypothesis that PLE1 and PLE6 hydrolyze endocannabinoids and promote inflammatory response. Consistent with the hypothesis, purified PLE1 and PLE6 efficaciously hydrolyzed 2-AG and AEA. PLE6 was 40-fold and 3-fold as active as PLE1 towards 2-AG and AEA, respectively. In addition, both PLE1 and PLE6 were highly sensitive to bis(4-nitrophenyl) phosphate (BNPP), an aryl phosphodiester known to predominately inhibit carboxylesterases. Based on the study with BNPP, PLEs contributed to the hydrolysis of 2-AG by 53.4 to 88.4% among various organs and cells. Critically, exogenous addition or transfection of PLE6 increased the expression and secretion of proinflammatory cytokines in response to the immunostimulant lipopolysaccharide (LPS). This increase was recapitulated in cocultured alveolar macrophages and PLE6 transfected cells in transwells. Finally, BNPP reduced inflammation trigged by LPS accompanied by reduced formation of AA and proinflammatory mediators. These findings define an innovative connection: PLE-endocannabinoid-inflammation. This mechanistic connection signifies critical roles of carboxylesterases in pathophysiological processes related to the metabolism of endocannabinoids.

DEC1 deficiency results in accelerated osteopenia through enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3ß signaling.

BACKGROUND: Human differentiated embryonic chondrocyte expressed gene 1 (DEC1) has been implicated in enhancing osteogenesis, a desirable outcome to counteract against deregulated bone formation such as retarded bone development, osteopenia and osteoporosis. METHODS AND RESULTS: DEC1 knockout (KO) and the age-matched wild-type (WT) mice were tested for the impact of DEC1 deficiency on bone development and osteopenia as a function of age. DEC1 deficiency exhibited retarded bone development at the age of 4 weeks and osteopenic phenotype in both 4- and 24-week old mice. However, the osteopenia was more severe in the 24-week age groups. Mechanistically, DEC1 deficiency downregulated the expression of bone-enhancing genes such as Runx2 and ß-catenin accompanied by upregulating DKK1, an inhibitor of the Wnt/ß-catenin signaling pathway. Consistently, DEC1 deficiency favored the attenuation of the integrated PI3KCA/Akt/GSK3ß signaling, a pathway targeting ß-catenin for degradation. Likewise, the attenuation was greater in the 24-week age group. These changes, however, were reversed by in vivo treatment with lithium chloride, a stabilizer of ß-catenin, and confirmed by gain-of-function study with DEC1 transfection into DEC1 KO bone marrow mesenchymal stem cells and loss-of-function study with siDEC1 lentiviral infection into the corresponding WT cells. CONCLUSION: DEC1 is a positive regulator with a broad activity spectrum in both bone development and maintenance, and the osteopenic phenotype accelerated by DEC1 deficiency is achieved by enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3ß signaling.

Remdesivir potently inhibits carboxylesterase-2 through covalent modifications: signifying strong drug-drug interactions.

Remdesivir was recently approved to treat COVID-19. While this antiviral agent delivers clinical benefits, several safety concerns in many cases have been raised. This study reports that remdesivir at nanomolar concentrations inhibits carboxylesterase-2 (CES2) through covalent modifications. CES2 is a major drug-metabolizing enzyme. The combination of high potency with irreversible inhibition concludes that cautions must be exercised when remdesivir is used along with drugs hydrolyzed by CES2.

Dechlorination and demethylation of ochratoxin A enhance blocking activity of PXR activation, suppress PXR expression and reduce cytotoxicity.

The pregnane X receptor (PXR) has been established to induce chemoresistance and metabolic diseases. Ochratoxin A (OTA), a mycotoxin, decreases the expression of PXR protein in human primary hepatocytes. OTA is chlorinated and has a methylated lactone ring. Both structures are associated with OTA toxicity. The study was to test the hypothesis that structural modifications differentially impact PXR blocking activity over cytotoxicity. To test this hypothesis, OTA-M and OTA-Cl/M were synthesized. OTA-M lacked the methyl group of the lactone-ring, whereas OTA-Cl/M had neither the methyl group nor the chlorine atom. The blocking activity of PXR activation was determined in a stable cell line, harboring both PXR (coding sequence) and its luciferase element reporter. OTA-Cl/M showed the highest blocking activity, followed by OTA-M and OTA. OTA-Cl/M was 60 times as potent as the common PXR blocker ketoconazole based on calculated IC50 values. OTA-Cl/M decreased by 90 % the expression of PXR protein and was the least cytotoxic among the tested compounds. Molecular docking identified that OTA and its derivatives interacted with different sets of residues in PXR, providing a molecular basis for selectivity. Excessive activation of PXR has been implicated in chemoresistance and metabolic diseases. Downregulation of PXR protein expression likely delivers an effective mechanism against structurally diverse PXR agonists.

Copper Sulfide Facilitates Hepatobiliary Clearance of Gold Nanoparticles through the Copper-Transporting ATPase ATP7B.

Metallic gold (Au) nanoparticles have great potential for a wide variety of biomedical applications. Yet, slow clearance of Au nanoparticles significantly hinders their clinical translation. Herein, we describe a strategy of utilizing the endogenous copper (Cu) clearance to improve the elimination of Au nanoparticles. Our mechanistic study reveals that a Cu-transporting P-type ATPase, ATP7B, mediates the exocytosis of CuS nanoparticles into bile canaliculi for their rapid hepatobiliary excretion. The efflux of CuS nanoparticles is adopted to facilitate the hepatobiliary clearance of Au nanoparticles through CuS-Au conjugation. Using two different CuS-Au nanoconjugates, we demonstrate that CuS increases the biliary Au excretion of CuS-Au nanospheres or CuS-Au nanorods in mice or rats in comparison to that of their respective unconjugated Au nanoparticles postintravenous injection. The current CuS-Au conjugation approach provides a feasible strategy to enhance the hepatobiliary clearance of Au nanoparticles that may be applicable to various structures.

Gemcitabine nanoparticles promote antitumor immunity against melanoma.

Myeloid-derived suppressor cells (MDSCs) promote tumor-mediated immunosuppression and cancer progression. Gemcitabine (Gem) is a MDSC-depleting chemotherapeutic agent; however, its clinical use is hampered by its drug resistance and inefficient in vivo delivery. Here we describe a strategy to formulate a Gem analogue gemcitabine monophosphate (GMP) into a lipid-coated calcium phosphate (LCP) nanoparticle, and investigate its antitumor immunity and therapeutic effects after systemic administrations. In the syngeneic mouse model of B16F10 melanoma, compared with free Gem, the LCP-formulated GMP (LCP-GMP) significantly induced apoptosis and reduced immunosuppression in the tumor microenvironment (TME). LCP-GMP effectively depleted MDSCs and regulatory T cells, and skewed macrophage polarization towards the antitumor M1 phenotype in the TME, leading to enhanced CD8+ T-cell immune response and profound tumor growth inhibition. Thus, engineering the in vivo delivery of MDSC-depleting agents using nanotechnology could substantially modulate immunosuppressive TME and boost T-cell immune response for enhanced antitumor efficacy.

17ß-estradiol suppresses carboxylesterases by activating c-Jun/AP-1 pathway in primary human and mouse hepatocytes.

In order to study the influence of estrogen on carboxylesterases, we investigated the effects of 17ß-estradiol on CES1 (Ces1d) and CES2 (Ces1e) in human and mouse hepatocytes. After being treated with 17ß-estradiol, the mRNA levels of CES1 and CES2 decreased by 29-39% and 28-55%, respectively, in the human hepatocytes from four donors. Consistently, the hydrolysis of para-nitrophenylacetate decreased markedly by 32% induced by 17ß-estradiol. Moreover, 17ß-estradiol decreased CES1 and CES2 by 45% and 47% respectively at protein levels. The response of altered expression of Ces1d (CES1) and Ces1e (CES2) to 17ß-estradiolin in mouse hepatocytes was very similar to that in the human hepatocytes. Further, the decreased Ces1d and Ces1e expression induced by 17ß-estradiol could be abolished by SP600125, an inhibitor of AP-1, both at mRNA and protein levels. Likewise, the increased c-Jun expression induced by 17ß-estradiol could almost be abolished by SP600125. In vivo, the expression of Ces1d, Ces1e and the hydrolytic activity of liver were higher in the ovariectomized female mice(OVX) than those in control mice(SHAM). However, when 17ß-estradiol was administrated, the expression of Ces1d, Ces1e and the hydrolytic activity of liver in the ovariectomized female mice (OVX+E2) became restored to their normal levels. Taken together, 17ß-estradiol suppresses carboxylesterases by activating c-Jun/AP-1 pathway in primary human and mouse hepatocytes. The findings can offer the potential gains in the safety and efficacy of pharmacotherapy for women, especially for pregnant and menopausal women.

Interleukin-6 Induces DEC1, Promotes DEC1 Interaction with RXRα and Suppresses the Expression of PXR, CAR and Their Target Genes.

Inflammatory burden is a primary cellular event in many liver diseases, and the overall capacity of drug elimination is decreased. PXR (pregnane X receptor) and CAR (constitutive androstane receptor) are two master regulators of genes encoding drug-metabolizing enzymes and transporters. DEC1 (differentiated embryonic chondrocyte-expressed gene 1) is a ligand-independent transcription factor and reportedly is induced by many inflammatory cytokines including IL-6. In this study, we used primary hepatocytes (human and mouse) as well as HepG2 cell line and demonstrated that IL-6 increased DEC1 expression and decreased the expressions of PXR, CAR, and their target genes. Overexpression of DEC1 had similar effect as IL-6 on the expression of these genes, and knockdown of DEC1 reversed their downregulation by IL-6. Interestingly, neither IL-6 nor DEC1 altered the expression of RXRα, a common dimerization partner for many nuclear receptors including PXR and CAR. Instead, DEC1 was found to interact with RXRα and IL-6 enhanced the interaction. These results conclude that DEC1 uses diverse mechanisms of action and supports IL-6 downregulation of drug-elimination genes and their regulators.