Bioreactor technologies to support liver function in vitro

Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drives efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models.National Institutes of Health (U.S.) (R01 EB010246)National Institutes of Health (U.S.) (P50-GM068762-08)National Institutes of Health (U.S.) (R01-ES015241)National Institutes of Health (U.S.) (P30-ES002109)5UH2TR000496-02National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (CBET-0939511)United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039

RhoA and DIAPH1 Mediate Adrenocorticotropin-Stimulated Cortisol Biosynthesis by Regulating Mitochondrial Trafficking

Steroid hormones are formed by the successive action of enzymes that are localized in mitochondria and the endoplasmic reticulum (ER). Compartmentalization of these enzymes in different subcellular organelles dictates the need for efficient transfer of intermediary metabolites between the mitochondrion and ER; however, the molecular determinants that regulate interorganelle substrate exchange are unknown. The objective of this study was to define the molecular mechanism by which adrenocorticotropin (ACTH) signaling regulates communication between mitochondria and the ER during steroidogenesis. Using live cell video confocal microscopy, we found that ACTH and dibutyryl cAMP rapidly increased the rate of mitochondrial movement. Inhibiting tubulin polymerization prevented both basal and ACTH/cAMP-stimulated mitochondrial trafficking and decreased cortisol secretion. This decrease in cortisol secretion evoked by microtubule inhibition was paralleled by an increase in dehydroepiandrosterone production. In contrast, treatment with paclitaxel to stabilize microtubules or latrunculin B to inhibit actin polymerization and disrupt microfilament organization increased both mitochondrial trafficking and cortisol biosynthesis. ACTH-stimulated mitochondrial movement was dependent on RhoA and the RhoA effector, diaphanous-related homolog 1 (DIAPH1). ACTH signaling temporally increased the cellular concentrations of GTP-bound and Ser-188 phosphorylated RhoA, which promoted interaction with DIAPH1. Expression of a dominant-negative RhoA mutant or silencing DIAPH1 impaired mitochondrial trafficking and cortisol biosynthesis and concomitantly increased the secretion of adrenal androgens. We conclude that ACTH regulates cortisol production by facilitating interorganelle substrate transfer via a process that is mediated by RhoA and DIAPH1, which act to coordinate the dynamic trafficking of mitochondria

CHARACTERIZATION OF CYP4A INDUCTION IN RAT LIVER BY INFLAMMATORY STIMULI: DEPENDENCE ON SEX, STRAIN, AND INFLAMMATION-EVOKED HYPOPHAGIA

This paper is available online at http://dmd.aspetjournals.org ABSTRACT: Acute treatment of rats with bacterial endotoxin or particulate irritants induces the expression of CYP4A mRNAs in rat liver and kidney. To determine whether all or part of these effects could be caused by hypophagia associated with the treatments, we pair-fed saline-injected rats to rats injected with endotoxin or the particulate irritant BaSO 4 . The effects of endotoxin on hepatic or renal CYP4A1, CYP4A2, or CYP4A3 expression 24 h after injection were clearly distinguishable in kinetics and magnitude from those of pair feeding, indicating that the effects of endotoxin are not caused by hypophagia. Conversely, BaSO 4 treatment caused a more profound hypophagia, and pair feeding to these animals produced effects similar to those of the irritant treatment, indicating that CYP4A induction by BaSO 4 is mainly caused by reduced food intake. To gain further insight into the mechanism of induction of CYP4A by these inflammatory agents, we studied the sex dependence of their effects in Fischer 344 and Sprague-Dawley rats. No significant strain differences were observed, but the induction of hepatic CYP4A mRNAs by endotoxin or BaSO 4 was either absent in females or significantly lower than in males. This sex specificity of induction of hepatic CYP4As has been reported previously for peroxisome proliferators, and thus our results are consistent with a role for the peroxisome proliferator-activated receptor-␣ in the induction of hepatic CYP4As by inflammatory agents

Steroidogenic Factor-1 and The Gonadotrope-Specific Element Enhance Basal and Pituitary Adenylate Cyclase-Activating Polypeptide-Stimulated Transcription of the Human Glycoprotein Hormone {alpha}-Subunit Gene in Gonadotropes

In the anterior pituitary, expression of the common glycoprotein hormone {alpha}-subunit ({alpha}GSU) is mediated in part by multiple response elements residing in the distal promoter (-435 bp). One such site is the gonadotrope-specific element (GSE), which is bound by the orphan nuclear receptor steroidogenic factor-1 (SF-1) and confers pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated {alpha}GSU expression. Here we investigated the functional importance of the GSE and SF-1 phosphorylation in both basal and stimulated {alpha}GSU transcription. Mutation of the GSE reduced basal and PACAP-stimulated {alpha}GSU promoter activity in the {alpha}T3-1 gonadotrope cell line. Overexpression of wild-type SF-1, but not an S203A mutant form of SF-1, enhanced basal and PACAP-stimulated {alpha}GSU promoter activity. The effect of PACAP on {alpha}GSU promoter activity was inhibited after overexpression of MAPK phosphatase. Helix assembly of the SF-1 ligand-binding domain was stimulated by PACAP in vitro via a MAPK-dependent pathway, as determined using a mammalian two-hybrid assay. PACAP quickly activated MAPK (within 5 min) and also resulted in elevated levels of phospho-cAMP response element-binding protein and phospho-SF-1, as judged by a specific antiphospho-S203 antibody; this effect was blocked by the MAPK kinase inhibitor, UO126. Collectively, these data demonstrate that SF-1 binds to the GSE and activates both basal and PACAP-stimulated {alpha}GSU transcription, which is further increased by phosphorylation at Ser203 via MAPK. These data suggest strongly that the induction of {alpha}GSU gene expression by peptide hormone signaling is coupled directly to the posttranslational status of SF-1