Molecular mechanisms controlling the phenotype and the EMT/MET dynamics of hepatocyte

The complex spatial and paracrine relationships between the various liver histotypes are essential for proper functioning of the hepatic parenchymal cells. Only within a correct tissue organization, in fact, they stably maintain their identity and differentiated phenotype. The loss of histotype identity, which invariably occurs in the primary hepatocytes in culture, or in vivo in particular pathological conditions (fibrosis and tumors), is mainly due to the phenomenon of epithelial-to-mesenchymal transition (EMT). The EMT process, that occurs in the many epithelial cells, appears to be driven by a number of general, non- tissue-specific, master transcriptional regulators. The reverse process, the mesenchymal-to epithelial transition (MET), as yet much less characterized at a molecular level, restores specific epithelial identities, and thus, must include tissue-specific master elements. In this review, we will summarize the so far unveiled events of EMT/MET occurring in liver cells. In particular, we will focus on hepatocyte and describe the pivotal role in the control of EMT/MET dynamics exerted by a tissue-specific molecular mini-circuitry. Recent evidence, indeed, highlighted as two transcriptional factors, the master gene of EMT Snail, and the master gene of hepatocyte differentiation HNF4α, exhorting a direct reciprocal repression, act as pivotal elements in determining opposite cellular outcomes. The different balances between these two master regulators, further integrated by specific microRNAs, in fact, were found responsible for the EMT/METs dynamics as well as for the preservation of both hepatocyte and stem/precursor cells identity and differentiation. Overall these findings impact the maintenance of stem cells and differentiated cells both in in vivo EMT/MET physio-pathological processes as well as in culture.The complex spatial and paracrine relationships between the various liver histotypes are essential for proper functioning of the hepatic parenchymal cells. Only within a correct tissue organization, in fact, they stably maintain their identity and differentiated phenotype. The loss of histotype identity, which invariably occurs in the primary hepatocytes in culture, or in vivo in particular pathological conditions (fibrosis and tumors), is mainly due to the phenomenon of epithelial-to-mesenchymal transition (EMT). The EMT process, that occurs in the many epithelial cells, appears to be driven by a number of general, non- tissue-specific, master transcriptional regulators. The reverse process, the mesenchymal-to epithelial transition (MET), as yet much less characterized at a molecular level, restores specific epithelial identities, and thus, must include tissue-specific master elements. In this review, we will summarize the so far unveiled events of EMT/MET occurring in liver cells. I

Childhood socioeconomic position and objectively measured physical capability levels in adulthood: a systematic review and meta-analysis

<p><b>Background:</b> Grip strength, walking speed, chair rising and standing balance time are objective measures of physical capability that characterise current health and predict survival in older populations. Socioeconomic position (SEP) in childhood may influence the peak level of physical capability achieved in early adulthood, thereby affecting levels in later adulthood. We have undertaken a systematic review with meta-analyses to test the hypothesis that adverse childhood SEP is associated with lower levels of objectively measured physical capability in adulthood.</p> <p><b>Methods and Findings:</b> Relevant studies published by May 2010 were identified through literature searches using EMBASE and MEDLINE. Unpublished results were obtained from study investigators. Results were provided by all study investigators in a standard format and pooled using random-effects meta-analyses. 19 studies were included in the review. Total sample sizes in meta-analyses ranged from N = 17,215 for chair rise time to N = 1,061,855 for grip strength. Although heterogeneity was detected, there was consistent evidence in age adjusted models that lower childhood SEP was associated with modest reductions in physical capability levels in adulthood: comparing the lowest with the highest childhood SEP there was a reduction in grip strength of 0.13 standard deviations (95% CI: 0.06, 0.21), a reduction in mean walking speed of 0.07 m/s (0.05, 0.10), an increase in mean chair rise time of 6% (4%, 8%) and an odds ratio of an inability to balance for 5s of 1.26 (1.02, 1.55). Adjustment for the potential mediating factors, adult SEP and body size attenuated associations greatly. However, despite this attenuation, for walking speed and chair rise time, there was still evidence of moderate associations.</p> <p><b>Conclusions:</b> Policies targeting socioeconomic inequalities in childhood may have additional benefits in promoting the maintenance of independence in later life.</p&gt

An epistatic mini-circuitry between the transcription factors Snail and HNF4\uce\ub1 controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs

Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal transition program, whereas maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular mini-circuitry between master elements of these biological processes might provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4\uce\ub1, directly represses the expression of the epithelial microRNAs (miRs)-200c and-34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4\uce\ub1, previously identified as a transcriptional repressor of Snail, induces the miRs-34a and-200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4\uce\ub1 and miRs-200a, b, c and-34a as epistatic elements controlling hepatic stem cell maintenance/differentiation. \uc2\ua9 2012 Macmillan Publishers Limited. All rights reserved

Two Isoforms of the mRNA Binding Protein IGF2BP2 Are Generated by Alternative Translational Initiation

IGF2BP2 is a member of a family of mRNA binding proteins that, collectively, have been shown to bind to several different mRNAs in mammalian cells, including one of the mRNAs encoding insulin-like growth factor-2. Polymorphisms in the Igf2bp2 gene are associated with risk of developing type 2 diabetes, but detailed functional characterisation of IGF2BP2 protein is lacking. By immunoblotting with C-terminally reactive antibodies we identified a novel IGF2BP2 isoform with a molecular weight of 58 kDa in both human and rodents, that is expressed at somewhat lower levels than the full-length 65 kDa protein. We demonstrated by mutagenesis that this isoform is generated by alternative translation initiation at the internal Met69. It lacks a conserved N-terminal RNA Recognition Motif (RRM) and would be predicted to differ functionally from the canonical full length isoform. We further investigated IGF2BP2 mRNA transcripts by amplification of cDNA using 5′-RACE. We identified multiple transcription start sites of the human, mouse and rat Igf2bp2 genes in a highly conserved region only 50–90 nts upstream of the major translation start site, ruling out the existence of N-terminally extended isoforms. We conclude that structural heterogeneity of IGF2BP2 protein should be taken into account when considering cellular function

Induction of Olig2+ Precursors by FGF Involves BMP Signalling Blockade at the Smad Level

During normal development oligodendrocyte precursors (OPCs) are generated in the ventral spinal cord in response to Sonic hedgehog (Shh) signalling. There is also a second, late wave of oligodendrogenesis in the dorsal spinal cord independent of Shh activity. Two signalling pathways, controlled by bone morphogenetic protein and fibroblast growth factor (FGF), are active players in dorsal spinal cord specification. In particular, BMP signalling from the roof plate has a crucial role in setting up dorsal neural identity and its inhibition is sufficient to generate OPCs both in vitro and in vivo. In contrast, FGF signalling can induce OPC production from dorsal spinal cord cultures in vitro. In this study, we examined the cross-talk between mitogen-activated protein kinase (MAPK) and BMP signalling in embryonic dorsal spinal cord cultures at the SMAD1/5/8 (SMAD1) transcription factor level, the main effectors of BMP activity. We have previously shown that FGF2 treatment of neural precursor cells (NPCs) derived from rat E14 dorsal spinal cord is sufficient to generate OPCs in vitro. Utilising the same system, we now show that FGF prevents BMP-induced nuclear localisation of SMAD1-phosphorylated at the C-terminus (C-term-pSMAD1). This nuclear exclusion of C-term-pSMAD1 is dependent on MAPK activity and correlates with OLIG2 upregulation, the obligate transcription factor for oligodendrogenesis. Furthermore, inhibition of the MAPK pathway abolishes OLIG2 expression. We also show that SMAD4, which acts as a common partner for receptor-regulated Smads including SMAD1, associates with a Smad binding site in the Olig2 promoter and dissociates from it upon differentiation. Taken together, these results suggest that FGF can promote OPC generation from embryonic NPCs by counteracting BMP signalling at the Smad1 transcription factor level and that Smad-containing transcriptional complexes may be involved in direct regulation of the Olig2 promoter

Impact of nine common type 2 diabetes risk polymorphisms in Asian Indian Sikhs: PPARG2 (Pro12Ala), IGF2BP2, TCF7L2 and FTO variants confer a significant risk

<p>Abstract</p> <p>Background</p> <p>Recent genome-wide association (GWA) studies have identified several unsuspected genes associated with type 2 diabetes (T2D) with previously unknown functions. In this investigation, we have examined the role of 9 most significant SNPs reported in GWA studies: [peroxisome proliferator-activated receptor gamma 2 (<it>PPARG2</it>; rs 1801282); insulin-like growth factor two binding protein 2 (<it>IGF2BP2</it>; rs 4402960); cyclin-dependent kinase 5, a regulatory subunit-associated protein1-like 1 (<it>CDK5</it>; rs7754840); a zinc transporter and member of solute carrier family 30 (<it>SLC30A8</it>; rs13266634); a variant found near cyclin-dependent kinase inhibitor 2A (<it>CDKN2A</it>; rs10811661); hematopoietically expressed homeobox (<it>HHEX</it>; rs 1111875); transcription factor-7-like 2 (<it>TCF7L2</it>; rs 10885409); potassium inwardly rectifying channel subfamily J member 11(<it>KCNJ11</it>; rs 5219); and fat mass obesity-associated gene (<it>FTO</it>; rs 9939609)].</p> <p>Methods</p> <p>We genotyped these SNPs in a case-control sample of 918 individuals consisting of 532 T2D cases and 386 normal glucose tolerant (NGT) subjects of an Asian Sikh community from North India. We tested the association between T2D and each SNP using unconditional logistic regression before and after adjusting for age, gender, and other covariates. We also examined the impact of these variants on body mass index (BMI), waist to hip ratio (WHR), fasting insulin, and glucose and lipid levels using multiple linear regression analysis.</p> <p>Results</p> <p>Four of the nine SNPs revealed a significant association with T2D; <it>PPARG2 </it>(Pro12Ala) [odds ratio (OR) 0.12; 95% confidence interval (CI) (0.03–0.52); p = 0.005], <it>IGF2BP2 </it>[OR 1.37; 95% CI (1.04–1.82); p = 0.027], <it>TCF7L2 </it>[OR 1.64; 95% CI (1.20–2.24); p = 0.001] and <it>FTO </it>[OR 1.46; 95% CI (1.11–1.93); p = 0.007] after adjusting for age, sex and BMI. Multiple linear regression analysis revealed significant association of two of nine investigated loci with diabetes-related quantitative traits. The 'C' (risk) allele of <it>CDK5 </it>(rs 7754840) was significantly associated with decreased HDL-cholesterol levels in both NGT (p = 0.005) and combined (NGT and T2D) (0.005) groups. The less common 'C' (risk) allele of <it>TCF7L2 </it>(rs 10885409) was associated with increased LDL-cholesterol (p = 0.010) in NGT and total and LDL-cholesterol levels (p = 0.008; p = 0.003, respectively) in combined cohort.</p> <p>Conclusion</p> <p>To our knowledge, this is first study reporting the role of some recently emerged loci with T2D in a high risk population of Asian Indian origin. Further investigations are warranted to understand the pathway-based functional implications of these important loci in T2D pathophysiology in different ethnicities.</p

Expression of MAGE-1 and -3 genes and gene products in human hepatocellular carcinoma

MAGE gene family encodes peptides recognized by autologous cytotoxic T lymphocytes in a major histocompatibility complex (MHC) class-I restricted fashion. In the present study, we have performed reverse-transcription polymerase chain reaction (RT-PCR) for the genes, as well as immunohistochemical analysis and Western blotting of MAGE-1 and -3 proteins in 33 surgically resected hepatocellular carcinomas (HCCs). MAGE-1 and -3 mRNAs were constitutively expressed exclusively in 78 and 42% of HCCs respectively. On immunohistochemistry with monoclonal antibodies, 77B for MAGE-1 and 57B for MAGE-3, MAGE-1 and -3 proteins were recognized in cytoplasm of only six among 33 (18%) and two of 29 HCCs (7%) respectively. The distribution pattern was mostly focal in HCC nodules. By contrast, the Western blot analysis revealed that the MAGE-1 (46 kDa) and -3 proteins (48 kDa) were expressed in 80 and 60% of 15 HCCs examined respectively. The proteins of MAGE-1 and -3 were also expressed exclusively in HCCs regardless of the histological grading and clinical staging. Our results indicate that the detection of the genes by RT-PCR or the proteins by Western blotting is useful for differentiating early HCCs from non-cancerous lesions, and that the peptides derived from MAGE-1 and -3 proteins might be suitable targets for immunotherapy of human HCC. © 1999 Cancer Research Campaig

On the Role of the Difference in Surface Tensions Involved in the Allosteric Regulation of NHE-1 Induced by Low to Mild Osmotic Pressure, Membrane Tension and Lipid Asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na+ for H+ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves

Mechanical Influences on Morphogenesis of the Knee Joint Revealed through Morphological, Molecular and Computational Analysis of Immobilised Embryos

Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint

MHC-peptide specificity and T-cell epitope mapping: where immunotherapy starts

The evaluation and characterization of epitope-specific human leukocyte antigen (HLA)-restricted memory T-cell reactivity is an important step for the development of preventive vaccines and peptide-based immunotherapies for viral and tumor diseases. The past decade has witnessed the use of HLA-restricted peptides as tools to activate strong immune responses of naive or memory T cells specifically. This has fuelled an active search for methodological approaches focusing on HLA and peptide associations. Here, we outline new perspective on the emerging opportunity of evaluating HLA and peptide restriction by using novel approaches, such as quantitative real-time PCR, that can identify epitope specificities that are potentially useful in clinical settings