Abstract Metabolic dysfunction‐associated steatohepatitis (MASH) is a leading cause of chronic liver disease with few therapeutic options. To narrow the translational gap in the development of pharmacological MASH treatments, a 3D liver model from primary human hepatocytes and non‐parenchymal cells derived from patients with histologically confirmed MASH was established. The model closely mirrors disease‐relevant endpoints, such as steatosis, inflammation and fibrosis, and multi‐omics analyses show excellent alignment with biopsy data from 306 MASH patients and 77 controls. By combining high‐content imaging with scalable biochemical assays and chemogenomic screening, multiple novel targets with anti‐steatotic, anti‐inflammatory, and anti‐fibrotic effects are identified. Among these, activation of the muscarinic M 1 receptor (CHRM1) and inhibition of the TRPM8 cation channel result in strong anti‐fibrotic effects, which are confirmed using orthogonal genetic assays. Strikingly, using biosensors based on bioluminescence resonance energy transfer, a functional interaction along a novel MASH signaling axis in which CHRM1 inhibits TRPM8 via G q/11 and phospholipase C‐mediated depletion of phosphatidylinositol 4,5‐bisphosphate can be demonstrated. Combined, this study presents the first patient‐derived 3D MASH model, identifies a novel signaling module with anti‐fibrotic effects, and highlights the potential of organotypic culture systems for phenotype‐based chemogenomic drug target identification at scale.
The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions -2762/-2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype.
Abstract Thi molecules involved in homotypic aggregation of the human Burkitt‐lymphoma cell line Raji were investigated by inhibition of reaggregation with carbohydrates and glycoconjugates, by inhibition of glycosylation, and enzyme treatment of the cell surface. Complete inhibition of reaggregation was achieved with bovine submaxillary mucin. Asialomucin, on the other hand, was not effective in this assay. Another potent inhibitor of reaggregation was the ganglioside GMI. The common carbohydrate structure of these molecules is NeuNAc‐(gal)‐galNAc. Lactosamine, fucosyllactosamine, sialyllactosamine, complex mannose type, or Thomsen‐Friedenreich antigen sequences are not involved in aggregation. Neuraminidase and chloroquine also abolished agglutination of cells. The finding that mucin, but not asialomucin, inhibits the reaction, demonstrates the importance of sialic acid in this process. Homotypic aggregation was shown to be resistant to trypsin. Using the glycosylation inhibitor tunicamycin we show that N‐glycosidically linked carbohydrate chains are involved in aggregation. Swainsonine or castanospermine, which inhibit processing of terminal sialyllactosamines to the mannose core, did not interfere with the reaction supporting the results of the inhibition assay. The data presented suggest the involvement of 2 molecules in homotypic aggregation of human Burkitt‐lymphoma cells. One component is a lectin‐like molecule containing N‐linked carbohydrate chains. The other component carries the neuraminidase‐sensitive and trypsin‐resistant determinant NeuNAc‐(gal)‐galNAc and, therefore, appears to be a glycolipid. This proposed lectin‐carbohydrate interaction in homotypic aggregation is further supported by the frequently observed dependence of lectins on divalent cations as indicated by inhibition of aggregation with EDTA and EGTA.
The nuclear receptor peroxisome proliferator–activated receptor (PPAR)α is known primarily as a regulator of fatty acid metabolism, energy balance, and inflammation, but evidence suggests a wider role in regulating the biotransformation of drugs and other lipophilic chemicals. We investigated whether PPARα directly regulates the transcription of cytochrome P450 3A4, the major human drug-metabolizing enzyme. Using chromatin immunoprecipitation in human primary hepatocytes as well as electrophoretic mobility shift and luciferase reporter-gene assays, we identified three functional PPARα-binding regions (PBR-I, -II, and -III) within ∼12 kb of the CYP3A4 upstream sequence. Furthermore, a humanized CYP3A4/3A7 mouse model showed in vivo induction of CYP3A4 mRNA and protein by [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY14,643) in liver but not in intestine, whereas hepatic occupancy of PBRs by PPARα was ligand independent. Using lentiviral gene knock-down and treatment with WY14,643 in primary human hepatocytes, PPARα was further shown to affect the expression of a distinct set of CYPs, including 1A1, 1A2, 2B6, 2C8, 3A4, and 7A1, but not 2C9, 2C19, 2D6, or 2E1. Interestingly, the common phospholipid 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-PC), previously proposed to reflect nutritional status and shown to be a specific endogenous ligand of PPARα, induced CYP3A4 (up to 4-fold) and other biotransformation genes in hepatocytes with similar selectivity and potency as WY14,643. These data establish PPARα as a direct transcriptional regulator of hepatic CYP3A4. This finding warrants investigation of both known and newly developed PPARα-targeted drugs for their drug-drug interaction potential. Furthermore, our data suggest that nutritional status can influence drug biotransformation capacity via endogenous phospholipid signaling.
Thomsen-Friedenreich (TF) antibodies were prepared from human serum by different enrichment procedures. This resulted in three antibody preparations all of which agglutinated neuraminidase-treated erythrocytes. On the other hand, each of the three antibody populations showed a distinct specificity pattern. Anti-TF1 antibodies could be inhibited in the hemagglutination inhibition assay by asialofetuin, asialotransferrin, asialoglycophorin and asialomucin. The sialylated form of these glycoproteins showed no inhibition. No significant inhibition could be achieved with several mono- or disaccharides. This suggests that anti-TF1 recognizes common structures on glycoproteins normally hidden by sialic acid. Anti-TF2 antibodies showed specificity for asialofetuin, bovine submaxillary mucin, asialomucin, asialoglycophorin, the disaccharide gal-beta (1-3)N-acetyl-galactosamine (galNAc) and nitrophenyl-beta-galactoside. Because asialotransferrin or unbound lactosamine were not inhibitory, we suppose that the residual common structure of the inhibitors is (gal)-galNAc-O-Ser/Thr, which is present in high amounts in submaxillary mucin. Anti-TF3 antibodies were inhibited by asialoglycophorin but not by asialomucin or asialofetuin. Strong saccharide inhibitors were gal-beta (1-3)galNAc, nitrophenyl-beta-galactoside, as well as galactose. Therefore, both of the antibody preparations, anti-TF2 and anti-TF3, could be inhibited by gal-beta-(1-3)galNAc, but showed preference to one or the other sugar component of the disaccharide resulting in a differential recognition of glycoconjugate inhibitors. Anti-TF2 and anti-TF3 seem to recognize the carbohydrates in the context of a protein backbone, because gal-beta-(1-3)galNAc in connection with a ceramide backbone (GM1) was not inhibitory. When tested on three human breast cancer cell lines, only anti-TF2 recognized epitopes exposed on the cell surface. We, therefore, conclude that human serum contains at least three subpopulations of TF antibodies with distinct specificities. Only anti-TF2 can detect cryptic erythrocyte epitopes which are also exposed on human breast cancer cell lines.
