Hippo signaling instructs ectopic but not normal organ growth.

The Hippo signaling pathway is widely considered a master regulator of organ growth because of the prominent overgrowth phenotypes caused by experimental manipulation of its activity. Contrary to this model, we show here that removing Hippo transcriptional output did not impair the ability of the mouse liver and Drosophila eyes to grow to their normal size. Moreover, the transcriptional activity of the Hippo pathway effectors Yap/Taz/Yki did not correlate with cell proliferation, and hyperactivation of these effectors induced gene expression programs that did not recapitulate normal development. Concordantly, a functional screen in Drosophila identified several Hippo pathway target genes that were required for ectopic overgrowth but not normal growth. Thus, Hippo signaling does not instruct normal growth, and the Hippo-induced overgrowth phenotypes are caused by the activation of abnormal genetic programs.

Functionality based method for simultaneous isolation of rodent hepatic sinusoidal cells.

Chronic liver disease is the result of long term exposure to viruses or toxins such as alcohol, fat and drugs, and forms the basis for the development of liver fibrosis and primary liver cancer. In vitro and in vivo models are key to study the pathways involved in chronic liver disease and for the development of therapeutics. 3D co-culture systems are becoming the in vitro standard, which requires freshly isolated primary hepatic cells. We developed a novel isolation method to simultaneously isolate liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs) and hepatic stellate cells (HSCs). The method exploits the scavenging activity of LSECs, the phagocytic capacity of KCs and the retinoid content of HSCs in vivo to enable direct processing by fluorescence-activated cell sorting without additional antibody binding and washing steps. UFACS3, for UV-FACS-based isolation of 3 non-parenchymal liver cell types, yields functional and pure LSECs (98 ± 1%), KCs (98 ± 1%) and HSCs (97 ± 3%), with less hands-on time from healthy and diseased rodent livers. This novel approach allows a fast and effective combined isolation of sinusoidal cells for further analysis.

The GALAD scoring algorithm based on AFP, AFP-L3, and DCP significantly improves detection of BCLC early stage hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of death in cirrhotic patients worldwide. The detection rate for early stage HCC remains low despite screening programs. Thus, the majority of HCC cases are detected at advanced tumor stages with limited treatment options. To facilitate earlier diagnosis, this study aims to validate the added benefit of the combination of AFP, the novel biomarkers AFP-L3, DCP, and an associated novel diagnostic algorithm called GALAD. Material and methods: Between 2007 and 2008 and from 2010 to 2012, 285 patients newly diagnosed with HCC and 402 control patients suffering from chronic liver disease were enrolled. AFP, AFP-L3, and DCP were measured using the µTASWako i30 automated immunoanalyzer. The diagnostic performance of biomarkers was measured as single parameters and in a logistic regression model. Furthermore, a diagnostic algorithm (GALAD) based on gender, age, and the biomarkers mentioned above was validated. Results: AFP, AFP-L3, and DCP showed comparable sensitivities and specifities for HCC detection. The combination of all biomarkers had the highest sensitivity with decreased specificity. In contrast, utilization of the biomarker-based GALAD score resulted in a superior specificity of 93.3 % and sensitivity of 85.6 %. In the scenario of BCLC 0/A stage HCC, the GALAD algorithm provided the highest overall AUROC with 0.9242, which was superior to any other marker combination. Conclusions: We could demonstrate in our cohort the superior detection of early stage HCC with the combined use of the respective biomarkers and in particular GALAD even in AFP-negative tumors.

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice.

BACKGROUND: Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. METHODS: Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-ß, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. RESULTS: OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-ß signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. CONCLUSIONS: OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

Syncoilin is an intermediate filament protein in activated hepatic stellate cells.

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, ß-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.

CTLA-4 interacts with STAT5 and inhibits STAT5-mediated transcription.

Cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152) is a member of the immunoglobulin gene superfamily with strong homology to the receptor CD28 with which it shares the ligands CD80 and CD86. Unlike CD28, a potent costimulator of T-cell responses, CTLA-4 is transiently expressed on the cell surface of activated T cells and appears to operate predominantly as a negative regulator of T-cell proliferation. Signal transduction mechanisms utilized by CTLA-4 remain unclear although several mechanisms have been implicated. In this study, we show that the cytoplasmic domain of CTLA-4, but not of CD28, binds to STAT5 in yeast two-hybrid assay and in coimmunoprecipitation assays. Mutations of Tyr165 and Tyr182 in CTLA-4 did not abrogate the interaction of STAT5 with CTLA-4. Finally, the overexpression of CTLA-4 in Jurkat T cells inhibits STAT-mediated activation of STAT5 responsive elements. These results suggest that CTLA-4 and STAT5 interact in T cells and that this interaction is important for CTLA-4 signalling.

Transforming growth factor beta signalling in vitro and in vivo: activin ligand-receptor interaction, Smad5 in vasculogenesis, and repression of target genes by the deltaEF1/ZEB-related SIP1 in the vertebrate embryo.

The identification and characterization of components of the transforming growth factor beta (TGFbeta) signalling pathway are proceeding at a very fast pace. To illustrate a number of our activities in this field, we first summarize our work aiming at the selection from a large collection of single residue substitution mutants of two activin A polypeptides in which D27 and K102, respectively, have been modified. This work has highlighted the importance of K102 and its positive charge for binding to activin type II receptors. Activin K102E, which did not bind to high-affinity receptor complexes, may be a valuable beta chain, when incorporated in recombinant inhibin to unambiguously detect novel inhibin binding sites at the cell surface. We then illustrate how Smad5 knockout mice and an overexpression approach with a truncated TGFbeta type II receptor in the mouse embryo can contribute to the identification of a novel TGFbeta-->TbetaRII/ALK1-->Smad5 pathway in endothelial cells in the embryo proper and the yolk sac vasculature. We conclude with a summary of our results with a Smad-interacting transcriptional repressor but focus on its biological significance in the vertebrate embryo.

SIP1 (Smad interacting protein 1) and deltaEF1 (delta-crystallin enhancer binding factor) are structurally similar transcriptional repressors.

BACKGROUND: Smad proteins are intracellular mediators of transforming growth factor-beta (TGFbeta) signalling that regulate gene expression by interacting with different classes of transcription factors including DNA-binding multi-zinc finger proteins. One of these, Smad interacting protein 1 (SIP1), is a novel two-handed zinc-finger protein that displays strong similarity with the transcriptional repressor delta-crystallin enhancer binding factor (deltaEF1). Here, we summarize what is known about the mechanism of action of both proteins and their role in vertebrate embryogenesis. Our data are discussed together with the present knowledge on other zinc-finger containing Smad interacting proteins. METHODS: The activities and function of SIP1 have been analysed through documentation of expression patterns, the effect of over-expression of SIP1 on target-gene expression, and promoter studies using Xenopus embryos. Moreover, S1P1/Smad complexes and their association with target promoter DNA were analyzed in biochemical studies. RESULTS: SIP1 is a transcriptional repressor displaying overlapping DNA binding specificities with deltaEF1. An in vivo target of SIP1 in Xenopus is a gene required for the formation of mesoderm, Brachyury (XBra). Our data indicate that SIP1 is required to confine XBra gene expression to the mesoderm. Furthermore, the expression pattern in Xenopus invites us to speculate that SIP1 plays a role in specification/differentiation of neuroectoderm. Unlike deltaEF1, SIP1 can bind directly to activated receptor regulated Smads (R-Smads) and recruit them to the DNA. This indicates that Smads may modulate the activity of SIP1 as a transcriptional repressor. CONCLUSIONS: Our data point to a role of SIP1 in developmental processes regulated by members of the TGFbeta family such as induction of mesoderm (mediated through activin-like signalling) and inhibition of neuroectoderm formation (mediated by bone morphogenetic proteins [BMPs]). Whereas SIP1 could act in TGFbeta signal transduction by virtue of interaction with activated R-Smads, genetic studies in the mouse indicate that deltaEF1 may act in certain TGFbeta pathways-i.e., BMPs and growth and differentiation factors (GDFs)-as well. The molecular mechanisms by which these transcriptional repressors act, as well as the function of the SIP1/Smad interaction, remain to be elucidated. CLINICAL RELEVANCE: Mutations in components of the TGFbeta signalling pathways have been associated with disease and congenital malformations. We anticipate that identification of Smad interacting transcription factors including SIP1 and their targets will help us to understand the molecular basis of certain pathologies.

XSIP1, a Xenopus zinc finger/homeodomain encoding gene highly expressed during early neural development.

We have isolated a Xenopus homologue of the zinc finger/homeodomain-containing transcriptional repressor Smad-interacting protein-1 (SIP1) from mouse. XSIP1 is activated at the early gastrula stage and transcription occurs throughout embryogenesis. At the beginning of gastrulation, XSIP1 is strongly expressed in prospective neurectoderm. At the neurula stage, XSIP1 is highly expressed within the neural plate but weakly in the dorsal midline. At later stages of development transcripts are detected primarily within the neural tube and neural crest. In the adult, XSIP1 expression is detected at variable levels in several organs.

Cooperation of Sp1 and p300 in the induction of the CDK inhibitor p21WAF1/CIP1 during NGF-mediated neuronal differentiation.

Addition of nerve growth factor (NGF) to PC12 cells promotes neuronal differentiation while inhibiting cell proliferation. In order to understand how NGF exerts its antimitogenic effect during differentiation, we have studied the mechanism by which this factor activates the promoter of the CDK inhibitor p21W4F1/CIP1. The minimal region of the p21 promoter required for the NGF-induction was mapped to a contiguous stretch of 10 bp located 83 bases upstream of the transcription initiation site. This GC-rich region was shown to interact specifically with the transcription factor Sp1 and the related protein Sp3, in either exponentially-growing or NGF-treated PC12 cells. The addition of NGF resulted in an accumulation of the transcriptional co-activator p300 in complexes associated with the NGF-responsive region. Transcriptional activity of Sp1, Sp3 and p300 was specifically induced by NGF in a Gal4-fusion assay, indicating that induction of p21 during neuronal differentiation may involve regulation of the activity of these factors by NGF. Furthermore, p300 was able to act as a co-activator for Sp1-mediated transcriptional activation in PC12 cells, suggesting that p300 and Sp1 may cooperate in activating p21 transcription during the withdrawal of neuronal precursors from the cell cycle. This hypothesis is supported by experiments showing that p300 and Sp1 form complexes in PC12 cells.

Cell cycle phase-specific surface expression of nerve growth factor receptors TrkA and p75(NTR).

Expression of the nerve growth factor (NGF) receptors TrkA and p75(NTR) was found to vary at the surface of PC12 cells in a cell cycle phase-specific manner. This was evidenced by using flow cytometric and microscopic analysis of cell populations labeled with antibodies to the extracellular domains of both receptors. Differential expression of these receptors also was evidenced by biotinylation of surface proteins and Western analysis, using antibodies specific for the extracellular domains of TrkA and p75(NTR). TrkA is expressed most strongly at the cell surface in M and early G1 phases, whereas p75(NTR) is expressed mainly in late G1, S, and G2 phases. This expression reflects the molecular and cellular responses to NGF in specific phases of the cell cycle; in the G1 phase NGF elicits both the anti-mitogenic effect, i.e., inhibition of the G1 to S transition, and the differentiation response whereas a survival effect is provoked elsewhere in the cell cycle. A model is proposed relating these responses to the surface expression of the two receptors. These observations open the way for novel approaches to the investigation of the mechanism of NGF signal transduction.

Distinct biological properties of two RET isoforms activated by MEN 2A and MEN 2B mutations.

Germline mutations of the RET proto-oncogene, which codes for a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). MEN 2 mutations have been shown to result in RET oncogenic activation. The RET gene encodes several isoforms whose biological properties, when altered by MEN 2 mutations, have not been thoroughly addressed yet. In this study, we have introduced a MEN 2A mutation (Cys634-->Arg) and the unique MEN 2B mutation (Met918-->Thr) in two RET isoforms of 1114 and 1072 amino acids which differ in the carboxy-terminus part. Herein, we report that each RET isoform activated by MEN 2A or MEN 2B mutation was transforming in fibroblasts and induced neuronal differentiation of pheochromocytoma PC12 cells. However, among the different RET-MEN 2 mutants, the long RET isoform activated by the MEN 2B mutation stimulated the most prominent neurite outgrowth in PC12 cells, while the short RET isoform counterpart elicited a very weak differentiation effect in PC12 cells. We further demonstrate that the morphological changes of PC12 cells caused by constitutively activated RET oncoproteins involved the engagement of a Ras-dependent pathway. These findings provide evidence that the biological properties of RET-MEN 2 mutants depend on the interplay between the RET isoforms and the nature of the activating MEN 2 mutation.

The CDK inhibitor p21WAF1/Cip1 is induced through a p300-dependent mechanism during NGF-mediated neuronal differentiation of PC12 cells.

The block of cell proliferation elicited by the addition of nerve growth factor (NGF) to exponentially-growing PC12 cells results, in part, from the inhibition of cyclin D1-associated kinase activity by p21WAF1/CIP1. NGF treatment of PC12 cells provokes the accumulation of p21 mRNA, due to transcriptional activation of the p21 promoter in a p53-independent manner. Transient expression of a mutated form of the adenovirus E1A protein (E1A dCR2), which retains its capacity to bind the transcriptional co-activator p300, completely abolishes the NGF-mediated stimulation of p21 promoter activity. This phenomenon can be reversed by ectopic expression of p300, suggesting that p300 is necessary for the induction of p21 by NGF. In addition, stable expression of E1A dCR2 in PC12 cells results in the inhibition of the NGF response, i.e. it prevents activation of the p21 promoter, cell cycle arrest, and neuronal differentiation. The signalling pathway from the TrkA receptor via the MAP kinase pathway is not altered in these cells. Together, these data indicate that p300 could play a pivotal role in the triggering of the anti-mitogenic effect of NGF and of neuronal differentiation.

Effect of nerve growth factor on the expression of cell cycle regulatory proteins in PC12 cells: dissection of the neurotrophic response from the anti-mitogenic response.

PC12 cells treated with nerve growth factor (NGF) undergo a G1 block and differentiate. Expression of selected cell cycle regulatory proteins was studied under culture conditions which permit observation of a differentiation response independently from a mitogenic or anti-mitogenic response. The expression of all cell cycle regulatory proteins studied is modulated by NGF addition to exponentially-growing cultures in the presence of serum. While levels of most of these proteins decrease, accumulation of cyclin D1 and the cyclin-dependent kinase inhibitor p21 Cip1/WAF1 is observed. Cyclin D1 associated kinase activity is inhibited, correlating with an increase in p21 protein. PC12 cells, synchronized by serum starvation, undergo morphological and functional differentiation in the presence of NGF. Neither cyclin D1 nor p21 are present in such cultures, nor is their expression upregulated by NGF, indicating that they are not required for this process. Removal of serum from differentiated PC12 cells results in loss of these proteins, but has no effect on differentiation or the nonproliferative state in presence of NGF. Together, the results indicate that cyclin D1 and p21 are not necessary for differentiation per se, nor are they required for maintenance of the differentiated state in the absence of serum.

Nerve growth factor-induced accumulation of PC12 cells expressing cyclin D1: evidence for a G1 phase block.

The anti-proliferative effect of nerve growth factor (NGF) on the rat pheochromocytoma cell line PC12 has been previously shown to be accompanied by the accumulation of cells in either the G1 phase with a 2c DNA content, or with a 4c DNA content characteristic for G2/M, as evidenced by flow cytometric analysis of DNA distribution using propidium iodide. Herein, these apparently conflicting results are clarified. The present studies indicate that a simple DNA distribution profile obtained by this technique can confound interpretation of the biological effects of NGF on cell-cycle distribution due to the presence of tetraploid cells. Using cyclin D1 and incorporation of bromodeoxyuridine as markers of respectively, G1 and S phase, we show that PC12 cultures can have a considerable amount of tetraploid cells which, when in the G1 phase, have a 4c DNA content and express cyclin D1. During exposure to NGF, this population increases, reflecting the accumulation of cells in the G1 phase of the cell cycle. The data presented, support the possibility that events affecting the expression or action of G1 regulatory proteins may be involved in the molecular mechanism of the anti-mitogenic effect of NGF.

Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells.

The expression of E and D-type cyclins, Cyclin-Dependent Kinase (CDK) 2 and 4, as well as CDK inhibitors p21Cip1 and p27Kip1 were examined during in vitro differentiation of mouse embryonic stem (ES) cells. ES cells cultured in presence of Differentiation Inhibitory Activity/Leukemia Inhibitory Factor (DIA/LIF) express very low levels of cyclin E/CDK2 complexes, p21Cip1 and p27Kip1 CDK inhibitors, while cyclin D/CDK4-associated kinase activity is undetectable. Withdrawal of DIA/LIF, which induces differentiation, results in the progressive up-regulation of all. Up-regulation of D cyclins occurs through an increase in the steady-state levels of mRNA, concomitantly with the activation of Brachyury and Goosecoid, two early markers of mesoderm differentiation. Similarly, cells from the epiblast of the early postimplantation mouse embryo do not express any cyclin D/CDK4 complexes. These are progressively upregulated at gastrulation and early organogenesis. DIA/LIF-stimulated ES cells are not growth-arrested by overexpression of p16Ink4a, a specific inhibitor of CDK4 and CDK6. We propose that the G1/S transition may be regulated by a minimal mechanism in mouse embryonic stem cells. Induction of differentiation triggers the establishment of a more sophisticated mechanism involving both cyclin D/CDK4- and CDK inhibitor-associated control of G1-phase progression.

Transcriptional regulation of tubulin gene expression in differentiating trochoblasts during early development of Patella vulgata.

The expression of alpha- and beta-tubulin genes during the early development of the marine mollusk Patella vulgata has been investigated. From the 32-cell stage onwards, an enhanced expression of both alpha- and beta-tubulin mRNAs was detected in the primary trochoblasts. After one additional cleavage, these cells become cleavage-arrested and then form cilia. They are the first cells to differentiate during Patella development. Later, alpha- and beta-tubulin mRNA is also found in the accessory and secondary trochoblasts. Together these three cell-lines form the prototroch, the ciliated locomotory organ of the trochophore larva. The early and abundant expression of tubulin genes precede and accompany cilia formation in the trochoblasts and provides us with an excellent molecular differentiation marker for these cells. Apart from the trochoblasts, tubulin gene expression was also found in other cells at some stages. At the 88-cell stage, elevated tubulin mRNA levels were found around the large nucleus of the mesodermal stem cell 4d. In later stages, tubulin gene expression was detected in the cells that form the flagella of the apical tuft and in the refractive bodies. An alpha-tubulin gene was isolated and characterized. A lacZ fusion gene under control of the 5' upstream region of this tubulin gene was microinjected into embryos at the two-cell stage. The reporter gene product was only detected in the three trochoblast cell-lines at the same time as tubulin genes were expressed in these cells. Reporter gene product was not detected in any other cells. Thus, this 5' upstream region of this alpha-tubulin gene contains all the elements required for the correct spatiotemporal pattern of expression.