The growth hormone-regulated transcription factors STAT5 and BCL6 coordinately regulate sex differences in mouse liver, primarily through effects in male liver, where male-biased genes are upregulated and many female-biased genes are actively repressed. Here we investigated whether CUX2, a highly female-specific liver transcription factor, contributes to an analogous regulatory network in female liver. Adenoviral overexpression of CUX2 in male liver induced 36% of female-biased genes and repressed 35% of male-biased genes. In female liver, CUX2 small interfering RNA (siRNA) preferentially induced genes repressed by adenovirus expressing CUX2 (adeno-CUX2) in male liver, and it preferentially repressed genes induced by adeno-CUX2 in male liver. CUX2 binding in female liver chromatin was enriched at sites of male-biased DNase hypersensitivity and at genomic regions showing male-enriched STAT5 binding. CUX2 binding was also enriched near genes repressed by adeno-CUX2 in male liver or induced by CUX2 siRNA in female liver but not at genes induced by adeno-CUX2, indicating that CUX2 binding is preferentially associated with gene repression. Nevertheless, direct CUX2 binding was seen at several highly female-specific genes that were positively regulated by CUX2, including A1bg, Cyp2b9, Cyp3a44, Tox, and Trim24. CUX2 expression and chromatin binding were high in immature male liver, where repression of adult male-biased genes and expression of adult femalebiased genes are common, suggesting that the downregulation of CUX2 in male liver at puberty contributes to the developmental changes establishing adult patterns of sex-specific gene expression. S ex differences in liver gene expression are widespread and affect a broad range of physiological processes, including steroid and drug metabolism, pheromone binding, and lipid metabolism. Hepatic sex-biased genes are regulated by growth hormone (GH) (35, 49), which is secreted by the pituitary gland in a sex-specific manner in rats, mice, and humans (16,28,44,53). Pituitary GH secretion is highly pulsatile in adult male rats and mice, where strong plasma peaks of GH are followed by periods when GH levels are below detection, whereas GH secretion is more frequent in females, resulting in a more continuous exposure to circulating GH. These sexually dimorphic plasma GH patterns stimulate sex differential patterns of tyrosine phosphorylation/activation and nuclear translocation of the transcription factors STAT5a and STAT5b (collectively, STAT5) (50). Thus, STAT5 activation is persistent in female liver but is intermittent in male liver, where it coincides with the onset of each plasma GH pulse (4, 43, 54). STAT5 positively regulates ϳ90% of male-biased genes and negatively regulates ϳ60% of female-biased genes in male mouse liver (5). STAT5 binding sites are found near 35 to 40% of sex-specific genes, suggesting they are directly regulated by STAT5 (54). However, a majority of sex-specific genes do not respond rapidly to GH-activated STAT5, suggesting indirect regulatory mechani...
Primary mouse hepatocytes are an important tool in the biomedical research field for the assessment of hepatocyte function. Several methods for hepatocyte isolation have been published; however, many of these methods require extensive handling and can therefore compromise the viability and function of the isolated cells. Since one advantage of utilizing freshly isolated cells is to maintain an environment in which the cells are more comparable to their in vivo state, it is important to have robust methods that produce cells with high viability, good purity and that function in a similar manner to that in their in vivo state. Here we describe a modified twostep method for the rapid isolation and characterization of mouse primary hepatocytes that results in high yields of viable cells. The asialoglycoprotein receptor (ASGPR), which is one of the most abundant cell surface receptors on hepatocytes, was used to monitor the function of the isolated hepatocytes by demonstrating specific binding of its ligand using a newly developed flow cytometry based ligand-receptor binding assay. Also, an in vitro screening method for siRNA drug candidates was successfully developed utilizing freshly isolated hepatocytes with minimum culture time.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Despite the prevalence of HCC, there is no effective, systemic treatment. The transcription factor LSF is a promising protein target for chemotherapy; it is highly expressed in HCC patient samples and cell lines, and promotes oncogenesis in rodent xenograft models of HCC. Here, we identify small molecules that effectively inhibit LSF cellular activity. The lead compound, factor quinolinone inhibitor 1 (FQI1), inhibits LSF DNA-binding activity both in vitro, as determined by electrophoretic mobility shift assays, and in cells, as determined by ChIP. Consistent with such inhibition, FQI1 eliminates transcriptional stimulation of LSF-dependent reporter constructs. FQI1 also exhibits antiproliferative activity in multiple cell lines. In LSF-overexpressing cells, including HCC cells, cell death is rapidly induced; however, primary or immortalized hepatocytes are unaffected by treatment with FQI1. The highly concordant structure–activity relationship of a panel of 23 quinolinones strongly suggests that the growth inhibitory activity is due to a single biological target or family. Coupled with the striking agreement between the concentrations required for antiproliferative activity (GI 50 s) and for inhibition of LSF transactivation (IC 50 s), we conclude that LSF is the specific biological target of FQIs. Based on these in vitro results, we tested the efficacy of FQI1 in inhibiting HCC tumor growth in a mouse xenograft model. As a single agent, tumor growth was dramatically inhibited with no observable general tissue cytotoxicity. These findings support the further development of LSF inhibitors for cancer chemotherapy.
Chronic inflammatory responses in the lung rely on the continual recruitment of leukocytes to the site of inflammation. Recent data have demonstrated a possible role for stromal cell-derived chemokines in leukocyte recruitment. In the present study we examined the production of interleukin (IL)-8 and ENA-78, members of the C-X-C family of chemokines, and macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, members of the C-C chemokine family, from pulmonary smooth muscle and endothelial cells. The production of IL-8 and ENA-78 was induced by early response cytokines, IL-1 and tumor necrosis factor (TNF), but not by immune-associated cytokines, IL-4, IL-10, or interferon (IFN)-gamma. In contrast, the production of MIP-1 alpha and MIP-1 beta by pulmonary vascular smooth muscle cells increased when stimulated by immune-associated cytokines as well as with IL-1 beta and TNF. The level of MIP-1 alpha production induced in smooth muscle cells by the immune-associated cytokines, IL-4, IFN-gamma, and IL-10 ranged from 0 to 340 pg/ml. The production of MIP-1 beta in response to the immune-associated cytokines IL-4, IFN-gamma, and IL-10 in smooth muscle cells ranged from 260 to 940 pg/ml. Human pulmonary artery endothelial cells did not generate MIP-1 alpha or MIP-1 beta in response to graded doses of any of the cytokines. These data demonstrate differential induction of C-X-C and C-C chemokines from nonimmune stromal cell populations.(ABSTRACT TRUNCATED AT 250 WORDS)
RNA interference (RNAi) has generated significant interest as a strategy to suppress viral infection, but in some cases antiviral activity of unmodified short-interfering RNA (siRNA) has been attributed to activation of innate immune responses. We hypothesized that immunostimulation by unmodified siRNA could mediate both RNAi as well as innate immune stimulation depending on the mode of drug delivery. We investigated the potential of immunostimulatory RNAs (isRNAs) to suppress influenza A virus in vivo in the mouse lung. Lipidoid 98N12-5(1) formulated with unmodified siRNA targeting the influenza nucleoprotein gene exhibited antiviral activity. Formulations were optimized to increase antiviral activity, but the antiviral activity of lipidoid-delivered siRNA did not depend on sequence homology to the influenza genome as siRNA directed against unrelated targets also suppressed influenza replication in vivo. This activity was primarily attributed to enhancement of innate immune stimulation by lipidoid-mediated delivery, which indicates increased toll-like receptor (TLR) activation by siRNA. Certain chemical modifications to the siRNA backbone, which block TLR7/8 activation but retain in vitro RNAi activity, prevented siRNA-mediated antiviral activity despite enhanced lipidoid-mediated delivery. Here, we demonstrate that innate immune activation caused by unmodified siRNA can have therapeutically relevant effects, and that these non-RNAi effects can be controlled through chemical modifications and drug delivery.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.