This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

Known as one of the hallmarks of cancer (Hanahan and Weinberg in Cell 100:57-70, 2000) cancer cell invasion of human body tissue is a complicated spatio-temporal multiscale process which enables a localised solid tumour to transform into a systemic, metastatic and fatal disease. This process explores and takes advantage of the reciprocal relation that solid tumours establish with the extracellular matrix (ECM) components and other multiple distinct cell types from the surrounding microenvironment. Through the secretion of various proteolytic enzymes such as matrix metalloproteinases or the urokinase plasminogen activator (uPA), the cancer cell population alters the configuration of the surrounding ECM composition and overcomes the physical barriers to ultimately achieve local cancer spread into the surrounding tissue. The active interplay between the tissue-scale tumour dynamics and the molecular mechanics of the involved proteolytic enzymes at the cell scale underlines the biologically multiscale character of invasion and raises the challenge of modelling this process with an appropriate multiscale approach. In this paper, we present a new two-scale moving boundary model of cancer invasion that explores the tissue-scale tumour dynamics in conjunction with the molecular dynamics of the urokinase plasminogen activation system. Building on the multiscale moving boundary method proposed in Trucu et al. (Multiscale Model Simul 11(1):309-335, 2013), the modelling that we propose here allows us to study the changes in tissue-scale tumour morphology caused by the cell-scale uPA microdynamics occurring along the invasive edge of the tumour. Our computational simulation results demonstrate a range of heterogeneous dynamics which are qualitatively similar to the invasive growth patterns observed in a number of different types of cancer, such as the tumour infiltrative growth patterns discussed in Ito et al. (J Gastroenterol 47:1279-1289, 2012).

burgdorferi will help to identify coregulated proteins that may cooperate to allow the organism to survive in a specific environment.

HNF1β is an atypical POU transcription factor that participates in a hierarchical network of transcription factors controlling the development and proper function of vital organs such as liver, pancreas, and kidney. Many inheritable mutations on HNF1β are the monogenic causes of diabetes and several kidney diseases. To elucidate the molecular mechanism of its function and the structural basis of mutations, we have determined the crystal structure of human HNF1β DNA binding domain in complex with a high-affinity promoter. Disease-causing mutations have been mapped to our structure, and their predicted effects have been tested by a set of biochemical/ functional studies. These findings together with earlier findings with a homologous protein HNF1α, help us to understand the structural basis of promoter recognition by these atypical POU transcription factors and the site-specific functional disruption by disease-causing mutations.HNF1β (hepatocyte nuclear factor 1β; also known as vHNF1 or TCF2) is a widely distributed transcription factor that plays a critical role in early vertebrate development and embryonic survival (1-3). First identified as a key regulator in the liver, HNF1β is also expressed in the pancreas, kidney, lung, ovary, testis, and throughout the gastrointestinal tract. In pancreatic β-cells, HNF1β is known to form an integrated regulatory network with other transcription factors such as HNF1α, HNF4α, Pdx-1, Foxa2, and NeuroD1 for organ development and proper function (1,4). Thus, in humans, heterozygous mutations in the HNF1β gene have been linked to neonatal diabetes (5) and the autosomal dominant subtype of diabetes known as MODY (maturity-onset diabetes of the young) (6). Extrapancreatic diseases are also increasingly recognized in different organs, especially in the kidney, with a variety of renal developmental disorders such as renal cysts, familial hypoplastic glomerulocystic kidney disease, renal malformation, and atypical familial hyperuricaemic nephropathy (7-11).POU transcription factors, which include Pit-1, Oct-1, and Unc-86 as founding members and are now expanded to more than 13 members in humans, are developmental regulators of various neuroendocrine organs, and their sequence-specific DNA binding is mediated by a bipartite motif that consists of a POU homeodomain (POU H ) and POU-specific domain (POU S ) (12,13). POU H is a 60 amino acid classic homeodomain made of three α-helices with the third as a DNA recognition helix, while POU S is an additional ∼75 amino acid all-α-helical motif that cooperates with POU H to enhance the binding affinity and specificity of DNA binding ( Figures 1 and 2) (12,14). HNF1α (MODY3 gene product, the most commonly mutated MODY protein) and HNF1β (MODY5 gene product) are atypical members of the POU transcription factors. Their POU S domains have at least one additional α-helix at the N-terminus, and the second † This work was funded by the Juvenile Diabetes Research Foundation (1-2004-506) (8,24). Recently, HNF1β has also been associated wi...

HNF4␣ (hepatocyte nuclear factor 4␣) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic ␤-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4␣ is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4␣ recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 Å crystal structure of human HNF4␣ DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1␣, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4␣ molecular function can cause significant effects in afflicted MODY patients.HNF4␣ is a novel member (NR2A1) of the nuclear receptor (NR) 2 family (1) and plays a crucial role in the development and function of vital organs. For example, HNF4␣ is essential for development of the liver (2), colon (3), and pancreas (4), and deletion of the HNF4␣ gene from the mouse genome results in embryonic lethality due to the failure to undergo normal gastrulation (5, 6). Conditional targeted gene disruption of HFN4␣ results in marked metabolic disregulation and increased mortality (7-9). In addition, HNF4␣ is known to activate a wide variety of genes involved in glucose, fatty acid, cholesterol, and amino acid metabolism in the liver, kidney, intestine, and pancreas (10 -12), including counterpart transcription factors, such as HNF1␣ (13), HNF6 (14), and pregnane X receptor (15), that in turn control additional liver and ␤-cell-specific target genes. Further underscoring the importance of HNF4␣ in pancreatic ␤-cells, mutations on HNF4␣ in humans are directly linked to the onset of MODY1 (maturity onset diabetes of the young 1) (Fig. 1A), one of the most common monogenic causes of diabetes, mainly characterized by impairment of glucosestimulated insulin secretion from the ␤-cells (16). A direct link between HNF4␣ and glucose-stimulated insulin secretion by pancreatic ␤-cells has been proven by cellular studies (17,18) and pancreatic ␤-cell-specific HNF4␣ knock-out mice (17). A recent genome-wide expression profiling study revealed that both HNF1␣ and HNF4␣ are the master regulators of the ␤-cells directly affecting their physiology (19,20). HNF4␣ is functionally very closely related to HNF1␣, since they crossregulate each other and form a common network of transcription factors that controls the development and function of hepatocyte and pancreatic islets (21, 22). A...

Hepatocyte nuclear factor 4 (HNF4) is a member of the nuclear receptor superfamily that plays a central role in organ development and metabolic functions. Mutations on HNF4 cause maturity-onset diabetes of the young (MODY), a dominant monogenic cause of diabetes. In order to understand the molecular mechanism of promoter recognition and the molecular basis of disease-causing mutations, the recombinant HNF4 DNA-binding domain was prepared and used in a study of its binding properties and in crystallization with a 21-mer DNA fragment that contains the promoter element of another MODY gene, HNF1. The HNF4 protein displays a cooperative and specific DNAbinding activity towards its target gene-recognition elements. Crystals of the complex diffract to 2.0 Å using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group C2, with unit-cell parameters a = 121. 63, b = 35.43, c = 70.99 Å , = 119.36 . A molecular-replacement solution has been obtained and structure refinement is in progress. This structure and the binding studies will provide the groundwork for detailed functional and biochemical studies of the MODY mutants.

Hepatocyte nuclear factor 1beta (HNF1beta) is a member of the POU transcription-factor family and binds the target DNA as a dimer with nanomolar affinity. The HNF1beta-DNA complex has been prepared and crystallized by hanging-drop vapor diffusion in 6%(v/v) PEG 300, 5%(w/v) PEG 8000, 8%(v/v) glycerol and 0.1 M Tris pH 8.0. The crystals diffracted to 3.2 A (93.9% completeness) using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group R3, with unit-cell parameters a = b = 172.69, c = 72.43 A. A molecular-replacement solution has been obtained and structure refinement is in progress. This structure will shed light on the molecular mechanism of promoter recognition by HNF1beta and the molecular basis of the disease-causing mutations found in it.

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