Identification of proteomic changes during human liver development by 2D-DIGE and mass spectrometry.

BACKGROUND/AIMS: The aim of this study was to identify human liver proteins that are associated with different stages of liver development. METHODS: We collected liver samples from 14 fetuses between 14 and 41 weeks of development, one child and four adults. Proteins which exhibited consistent and significant variations during development by two-dimensional differential in gel electrophoresis (2D-DIGE) were subjected to peptide mass fingerprint analysis by MALDI-TOF mass spectrometry. Real-time PCR analysis confirmed, at the transcriptional level, the data obtained by the proteomic approach. RESULTS: Among a total of 80 protein spots showing differential expression, we identified 42 different proteins or polypeptide chains, of which 26 were upregulated and 16 downregulated in developing in comparison to adult liver. These proteins could be classified in specific groups according to their function. By comparing their temporal expression profiles, we identified protein groups that were associated with different developmental stages of human fetal liver and suggest that the changes in protein expression observed during the 20- to 36-week time window play a pivotal role in liver development. CONCLUSIONS: The identification of these proteins may represent good markers of human liver and stem cells differentiation.

Possible involvement of pregnane X receptor-enhanced CYP24 expression in drug-induced osteomalacia.

Vitamin D controls calcium homeostasis and the development and maintenance of bones through vitamin D receptor activation. Prolonged therapy with rifampicin or phenobarbital has been shown to cause vitamin D deficiency or osteomalacia, particularly in patients with marginal vitamin D stores. However, the molecular mechanism of this process is unknown. Here we show that these drugs lead to the upregulation of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) gene expression through the activation of the nuclear receptor pregnane X receptor (PXR; NR1I2). CYP24 is a mitochondrial enzyme responsible for inactivating vitamin D metabolites. CYP24 mRNA is upregulated in vivo in mice by pregnenolone 16alpha-carbonitrile and dexamethasone, 2 murine PXR agonists, and in vitro in human hepatocytes by rifampicin and hyperforin, 2 human PXR agonists. Moreover, rifampicin increased 24-hydroxylase activity in these cells, while, in vivo in mice, pregnenolone 16alpha-carbonitrile increased the plasma concentration of 24,25-dihydroxyvitamin D(3). Transfection of PXR in human embryonic kidney cells resulted in rifampicin-mediated induction of CYP24 mRNA. Analysis of the human CYP24 promoter showed that PXR transactivates the sequence between -326 and -142. We demonstrated that PXR binds to and transactivates the 2 proximal vitamin D-responsive elements of the human CYP24 promoter. These data suggest that xenobiotics and drugs can modulate CYP24 gene expression and alter vitamin D(3) hormonal activity and calcium homeostasis through the activation of PXR.

Fatty Acid composition and levels of selected polyunsaturated Fatty acids in four commercial important freshwater fish species from lake victoria, Tanzania.

Fatty acids (FAs) particularly ω3 and ω6 polyunsaturated fatty acids (PUFAs) play important role in human health. This study aimed to investigate the composition and levels of selected ω3 PUFAs in four commercial fish species, Nile perch (Lates niloticus), Nile tilapia (Oreochromis niloticus), Tilapia zillii, and dagaa (Rastrineobola argentea) from Mwanza Gulf in Lake Victoria. The results indicated that 36 types of FAs with different saturation levels were detected. These FAs were dominated by docosahexaenoic (DHA), eicosapentaenoic (EPA), docosapentaenoic (DPA), and eicosatetraenoic acids. O. niloticus had the highest composition of FAs (34) compared to L. niloticus (27), T. zillii (26), and R. argentea (21). The levels of EPA differed significantly among the four commercial fish species (F = 6.19, P = 0.001). The highest EPA levels were found in R. argentea followed by L. niloticus and O. niloticus and the lowest in T. zillii. The DPA levels showed no significant difference among the four fish species studied (F = 0.652, P = 0.583). The study concluded that all four commercial species collected from Mwanza Gulf are good for human health, but R. argentea is the best for consumption because it contains higher levels of ω3 FAs, mainly EPA.

Differential regulation of constitutive androstane receptor expression by hepatocyte nuclear factor4alpha isoforms.

Constitutive androstane receptor (CAR; NR1I3) controls the metabolism and elimination of endogenous and exogenous toxic compounds by up-regulating a battery of genes. In this work, we analyzed the expression of human CAR (hCAR) in normal liver during development and in hepatocellular carcinoma (HCC) and investigated the effect of hepatocyte nuclear factor 4alpha isoforms (HNF4alpha1 and HNF4alpha7) on the hCAR gene promoter. By performing functional analysis of hCAR 5'-deletions including mutants, chromatin immunoprecipitation in human hepatocytes, electromobility shift and cotransfection assays, we identified a functional and species-conserved HNF4alpha response element (DR1: ccAGGCCTtTGCCCTga) at nucleotide -144. Both HNF4alpha isoforms bind to this element with similar affinity. However, HNF4alpha1 strongly enhanced hCAR promoter activity whereas HNF4alpha7 was a poor activator and acted as a repressor of HNF4alpha1-mediated transactivation of the hCAR promoter. PGC1alpha stimulated both HNF4alpha1-mediated and HNF4alpha7-mediated hCAR transactivation to the same extent, whereas SRC1 exhibited a marked specificity for HNF4alpha1. Transduction of human hepatocytes by HNF4alpha7-expressing lentivirus confirmed this finding. In addition, we observed a positive correlation between CAR and HNF4alpha1 mRNA levels in human liver samples during development, and an inverse correlation between CAR and HNF4alpha7 mRNA levels in HCC. These observations suggest that HNF4alpha1 positively regulates hCAR expression in normal developing and adult livers, whereas HNF4alpha7 represses hCAR gene expression in HCC.

Ascidians as a vertebrate-like model organism for physiological studies of Rho GTPase signaling.

GTPases of the Rho family are evolutionarily conserved proteins that control cell shape dynamics during physiological processes as diverse as cell migration and polarity, axon outgrowth and guidance, apoptosis and phagocytosis. In mammals, 18 Rho proteins are distributed in 7 subfamilies. Rho, Rac and Cdc42 are the best-characterized ones, benefiting from the use of worm and drosophila, which only express these 3 subfamilies. An additional model would therefore help understand the physiological role of other mammalian subfamilies. We identified in genome databases the complete Rho family of two ascidians, Ciona intestinalis and Ciona savignyi, and showed that these families contain single ancestors of most mammalian Rho subfamilies. In Ciona intestinalis, all Rho genes are expressed and display specific developmental variations of mRNA expression during tadpole formation. Although C. intestinalis expresses five additional Rac compared to the closely related Ciona savignyi, only two appeared fully active in functional assays. Last, we identified in Ciona intestinalis database more than 50 Rho regulators (RhoGEFs and RhoGAPs) and 20 effector targets, whose analysis further supports the notion that Rho signaling components are of comparable complexity in mammals and ascidians. Since the tadpole of ascidians combines vertebrate-like developmental features with reduced cell number, particularly adapted to evolutionary and developmental biology studies, our data advocate this model for physiological studies of Rho signaling pathways.