Mary C. Weiss

Identification of the CC chemokines TARC and macrophage inflammatory protein-1β as novel functional ligands for the CCR8 receptor

Chemokines are key molecules in directing leukocyte migration toward sites of inflammation. We have previously cloned a putative CC chemokine receptor gene, TER1, whose expression is restricted to lymphoid tissues and cell lines. Recently, this receptor has been shown to signal in response to the human CC chemokine I‐309 and thus it has been renamed CCR8 according to the current nomenclature. In the present study, we report the identification of the CC chemokines thymus and activation‐regulated cytokine (TARC) and macrophage inflammatory protein‐1β (MIP‐1β) as CCR8 ligands, as they induce chemotaxis in CCR8 Jurkat stable transfectants. Furthermore, we have generated a polyclonal antiserum that is able to recognize the CCR8 molecule in transfectant lysates. The pattern of CCR8 mRNA expression and the functional effects exerted by its ligand suggest that the triggering of this receptor may regulate multiple functions including activation, migration and proliferation of lymphoid cells.

Leukocystatin, A New Class II Cystatin Expressed Selectively by Hematopoietic Cells

We describe a new cystatin in both mice and humans, which we termed leukocystatin. This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions. As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs. In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns. The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAG™ fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins. The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system.

The activity of the highly inducible mouse phenylalanine hydroxylase gene promoter is dependent upon a tissue-specific, hormone-inducible enhancer.

Expression of the phenylalanine hydroxylase gene in livers and kidneys of rodents is activated at birth and is induced by glucocorticoids and cyclic AMP in the liver. Regulatory elements in a 10-kb fragment upstream of the mouse gene have been characterized. The promoter lacks TAATA and CCAAT consensus sequences and shows only extremely weak activity in transitory expression assays with phenylalanine hydroxylase-producing hepatoma cells. No key elements for regulation of promoter activity are localized within 2 kb of upstream sequences. However, a liver-specific DNase I-hypersensitive site at kb -3.5 comprises a tissue-specific and hormone-inducible enhancer. This enhancer contains multiple protein binding sites, including sites for ubiquitous factors (NF1 and AP1), the glucocorticoid receptor, and the hepatocyte-enriched transcription factors hepatocyte nuclear factor 1 (HNF1) and C/EBP. Mutation revealed that the last two sites are critical not only for basal activity but also for obtaining a maximal hormone response. Efficient transcription from the highly inducible promoter shows absolute dependence upon the enhancer at kb - 3.5, which in turn requires HNF1 and C/EBP as well as hormones. The regulatory region of the mouse phenylalanine hydroxylase gene differs totally from that of humans, even though the genes of both species are expressed essentially in the liver. Furthermore, the phenylalanine hydroxylase gene of mice shows an expression pattern very similar to those of the rodent tyrosine aminotransferase and phosphoenolpyruvate carboxykinase genes, yet each shows a different organization of its regulatory region.

Transcription factor HNF-6/OC-1 inhibits the stimulation of the HNF-3α/Foxa1 gene by TGF-β in mouse liver

A network of liver‐enriched transcription factors controls differentiation and morphogenesis of the liver. These factors interact via direct, feedback, and autoregulatory loops. Previous work has suggested that hepatocyte nuclear factor (HNF)‐6/OC‐1 and HNF‐3α/FoxA1 participate coordinately in this hepatic network. We investigated how HNF‐6 controls the expression of Foxa1. We observed that Foxa1 expression was upregulated in the liver of Hnf6−/− mouse embryos and in bipotential mouse embryonic liver (BMEL) cell lines derived from embryonic Hnf6−/− liver, suggesting that HNF‐6 inhibits the expression of Foxa1. Because no evidence for a direct repression of Foxa1 by HNF‐6 was found, we postulated the existence of an indirect mechanism. We found that the expression of a mediator and targets of the transforming growth factor beta (TGF‐β) signaling was increased both in Hnf6−/− liver and in Hnf6−/− BMEL cell lines. Using these cell lines, we demonstrated that TGF‐β signaling was increased in the absence of HNF‐6, and that this resulted from upregulation of TGF‐β receptor II expression. We also found that TGF‐β can stimulate the expression of Foxa1 in Hnf6+/+ cells and that inhibition of TGF‐β signaling in Hnf6−/− cells down‐regulates the expression of Foxa1. In conclusion, we propose that Foxa1 upregulation in the absence of HNF‐6 results from increased TGF‐β signaling via increased expression of the TGF‐β receptor II. We further conclude that HNF‐6 inhibits Foxa1 by inhibiting the activity of the TGF‐β signaling pathway. This identifies a new mechanism of interaction between liver‐enriched transcription factors whereby one factor indirectly controls another by modulating the activity of a signaling pathway. (HEPATOLOGY 2004;40:1266–1274.)

Phenobarbital, dexamethasone and benzanthracene induce several cytochrome P450 mRNAs in rat hepatoma cells

Hepatoma cells derived from the Reuber H35 rat hepatoma express cytochrome P450 enzymes of two major families: polycyclic aromatic hydrocarbon‐inducible forms are found in both differentiated and dedifferentiated cells while phenobarbital (PB)‐inducible forms are found only in differentiated cells. We report here that (i) benzanthracene and PB induce P450 c mRNA in differentiated and dedifferentiated cells and (ii) dexamethasone and PB induce P450 b/e and/or P450 PB1 mRNAs in differentiated cells but not in dedifferentiated cells.

In vivo role of the HNF4α AF‐1 activation domain revealed by exon swapping

The gene encoding the nuclear receptor hepatocyte nuclear factor 4α (HNF4α) generates isoforms HNF4α1 and HNF4α7 from usage of alternative promoters. In particular, HNF4α7 is expressed in the pancreas whereas HNF4α1 is found in liver, and mutations affecting HNF4α function cause impaired insulin secretion and/or hepatic defects in humans and in tissue‐specific ‘knockout’ mice. HNF4α1 and α7 isoforms differ exclusively by amino acids encoded by the first exon which, in HNF4α1 but not in HNF4α7, includes the activating function (AF)‐1 transactivation domain. To investigate the roles of HNF4α1 and HNF4α7 in vivo, we generated mice expressing only one isoform under control of both promoters, via reciprocal swapping of the isoform‐specific first exons. Unlike Hnf4α gene disruption which causes embryonic lethality, these ‘α7‐only’ and ‘α1‐only’ mice are viable, indicating functional redundancy of the isoforms. However, the former show dyslipidemia and preliminary results indicate impaired glucose tolerance for the latter, revealing functional specificities of the isoforms. These ‘knock‐in’ mice provide the first test in vivo of the HNF4α AF‐1 function and have permitted identification of AF‐1‐dependent target genes.

Embryonic liver cells and permanent lines as models for hepatocyte and bile duct cell differentiation

Analysis of liver cells during development is facilitated by the possibility of complementing in vivo analysis with experiments on cultured cells. In this review, we discuss results from several laboratories concerning bipotential hepatic stem cells from mouse (HBC-3, H-CFU-C, MMH and BMEL), rat (rhe14321) and primate (IPFLS) embryos. Several groups have used fluorescence-activated cell sorting to identify clonogenic bipotential cells; others have derived bipotential cell lines by plating liver cell suspensions and cloning. The bipotential cells, which probably originate from hepatoblasts, can differentiate as hepatocytes or bile duct cells, and undergo morphogenesis in culture. Disparities in differentiation can be explained by distinct medium compositions, extracellular matrix coated culture surfaces, and gene expression detection methods. Potential applications of these cell lines are discussed.

Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers

The ability to purify to homogeneity a population of hepatic progenitor cells from adult liver is critical for their characterization prior to any therapeutic application. As a step in this direction, we have used a bipotential liver cell line from 14 days postcoitum mouse embryonic liver to compile a list of cell surface markers expressed specifically by liver progenitor cells. These cells, known as bipotential mouse embryonic liver (BMEL) cells, proliferate in an undifferentiated state and are capable of differentiating into hepatocyte‐like and cholangiocyte‐like cells in vitro. Upon transplantation, BMEL cells are capable of differentiating into hepatocytes and cholangiocytes in vivo. Microarray and Gene Ontology (GO) analysis of gene expression in the 9A1 and 14B3 BMEL cell lines grown under proliferating and differentiating conditions was used to identify cell surface markers preferentially expressed in the bipotential undifferentiated state. This analysis revealed that proliferating BMEL cells express many genes involved in cell cycle regulation, whereas differentiation of BMEL cells by cell aggregation causes a switch in gene expression to functions characteristic of mature hepatocytes. In addition, microarray data and protein analysis indicated that the Notch signaling pathway could be involved in maintaining BMEL cells in an undifferentiated stem cell state. Using GO annotation, a list of cell surface markers preferentially expressed on undifferentiated BMEL cells was generated. One marker, Cd24a, is specifically expressed on progenitor oval cells in livers of diethyl 1,4‐dihydro‐2,4,6‐trimethyl‐3,5‐pyridinedicarboxylate‐treated animals. We therefore consider Cd24a expression a candidate molecule for purification of hepatic progenitor cells.

Intracellular Targeting of the Type-Iα Regulatory Subunit of cAMP-Dependent Protein Kinase

Abstract The specificity of cyclic adenosine monophosphate (cAMP)-mediated signaling events is achieved by the composition and biochemical properties of the different cAMP-dependent protein kinase holoenzymes (PKAI and II) and by compartmentalization of PKA to discrete subcellular locations. Intracellular localization is mediated by interaction with A-kinase anchoring proteins (AKAPs) that recruit PKAII close to its substrates and to sites where it can respond optimally to local changes in intracellular cAMP concentration, thereby directing and amplifying the effects of cAMP. This review presents recent evidence that indicates that specific AKAPs mediate PKAI anchoring through interaction with its regulatory subunit RIα, notably at the neuromuscular junction of skeletal muscle.

Alternative 5′-exons of the mouse cAMP-dependent protein kinase subunit RIα gene are conserved and expressed in both a ubiquitous and tissue-restricted fashion

The activity of cAMP‐dependent protein kinase is controlled by its regulatory subunits. Mouse RIα regulatory subunit expression is initiated from five different non‐coding 5′‐regions (exons 1a, 1b, 1c, 1d and 1e). This organization appears to be conserved among species. All mouse tissues accumulate exon 1a and 1b transcripts and most contain more 1b than 1a, except brain, heart and oesophagus. Exon 1d and 1e transcripts are found in several tissues, while exon 1c is testis‐specific. All five transcripts are in RIα‐rich tissues: gonads and adrenal glands.