Haploinsufficiency of Def Activates p53-Dependent TGFβ Signalling and Causes Scar Formation after Partial Hepatectomy

The metazoan liver exhibits a remarkable capacity to regenerate lost liver mass without leaving a scar following partial hepatectomy (PH). Whilst previous studies have identified components of several different signaling pathways that are essential for activation of hepatocyte proliferation during liver regeneration, the mechanisms that enable such regeneration to occur without accompanying scar formation remain poorly understood. Here we use the adult zebrafish liver, which can regenerate within two weeks following PH, as a new genetic model to address this important question. We focus on the role of Digestive-organ-expansion-factor (Def), a nucleolar protein which has recently been shown to complex with calpain3 (Capn3) to mediate p53 degradation specifically in the nucleolus, in liver regeneration. Firstly, we show that Def expression is up-regulated in the wild-type liver following amputation, and that the def^hi429/+ heteroozygous mutant (def^+/−) suffers from haploinsufficiency of Def in the liver. We then show that the expression of pro-inflammatory cytokines is up-regulated in the def^+/− liver, which leads to distortion of the migration and the clearance of leukocytes after PH. Transforming growth factor β (TGFβ) signalling is thus activated in the wound epidermis in def^+/− due to a prolonged inflammatory response, which leads to fibrosis at the amputation site. Fibrotic scar formation in def^+/− is blocked by the over-expression of Def, by the loss-of-function of p53, and by treatment with anti-inflammation drug dexamethasone or TGFβ-signalling inhibitor SB431542. We finally show that the Def- p53 pathway suppresses fibrotic scar formation, at least in part, through the regulation of the expression of the pro-inflammatory factor, high-mobility group box 1. We conclude that the novel Def- p53 nucleolar pathway functions specifically to prevent a scar formation at the amputation site in a normal amputated liver.     

Def Functions as a Cell Autonomous Factor in Organogenesis of Digestive Organs in Zebrafish

Digestive organs originate from the endoderm. Morphogenesis of the digestive system is precisely controlled by multiple factors that dictate the cell fate and behavior so that the specific digestive organs are timely formed in the right place and develop into right size and structure. We showed previously that digestive organ expansion factor (def) is a gene whose expression is enriched in the liver, pancreas and intestine. Loss-of-function of def in the def^hi429 mutant confers hypoplastic digestive organs partly due to alteration of expression of genes related to the p53 pathway. However, the molecular mechanism for the involvement of Def in the organogenesis of digestive organs is still largely unknown. For example, it is not known whether Def regulates specific pathways in a specific organ. To address this question, we generated four independent Tg(fabp10a:def) transgenic fish lines which over-expressed Def specifically in the liver. We characterized Tg-I, one of the transgenic lines, in detail with genetic, molecular and histological approaches. We found that Tg-I restored the liver but not exocrine pancreas and intestine development in the def^hi429 mutant. However, Tg-I adult fish in the wild type (WT) background exhibits reduced liver-to-body ratio and all four transgenic lines conferred abnormal intrahepatic structure. Microarray data analysis showed that certain specific functional pathways were affected in the liver of Tg-I. These results demonstrate that Def functions in a cell autonomous manner during early liver development and aberrant Def protein expression might lead to disruption of the structural integrity of a normal adult liver.     

Generation of mice with hepatocyte-specific conditional deletion of sphingosine kinase 1

SphK1 gene has different roles in various types of cells in liver diseases, but most studies are based on global knockout mice, which hampers the study on the cellular and molecular mechanisms of SphK1. In order to further study the role of SphK1 in liver, SphK1 conditional knockout mice were constructed. A liver-specific SphK1 gene knockout mouse model was constructed by the Cre/Loxp recombinant enzyme system. PCR technologies and western blotting were used to identified the elimination of SphK1 gene in hepatocytes. SphK1^flox/flox mice were used as a control group to verify the effectiveness of SphK1 liver-specific knockout mice from the profile, pathology, and serology of mice. The ablation of SphK1 in hepatic parenchymal cells was demonstrated by fluorescent in situ hybridization and the contents of S1P and Sph were measured by ELISA kit. The genotypes of liver in SphK1 conditional knockout mice were different from that of other organs. The mRNA and protein levels of SphK1 in liver tissue of SphK1 conditional knockout mice were almost depleted by compared with SphK1^flox/flox mice. Physiology and pathology showed no significant difference between SphK1 liver conditional knockout mice and SphK1^flox/flox mice. Additionally, SphK1 was eliminated in hepatocytes, leading to the reduce of S1P content in hepatocytes and liver tissues and the increase of Sph content in hepatocytes. The model of SphK1 gene liver conditional knockout mice was successfully constructed, providing a tool for the study of the roles of SphK1 in hepatocyte and liver diseases.

     

Ribosome biogenesis protein Urb1 acts downstream of mTOR complex 1 to modulate digestive organ development in zebrafish

Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in zebrafish urb1, which encodes an essential ribosome biogenesis protein. The urb1 cq31 mutant exhibits hypoplastic digestive organs, which is caused by impaired cell proliferation with the differentiation of digestive organ progenitors unaffected. Knockdown of mtor or raptor leads to similar hypoplastic phenotypes and reduced expression of urb1 in the digestive organs. Overexpression of Urb1 results in overgrowth of digestive organs, and can efficiently rescue the hypoplastic liver and pancreas in the mtor and raptor morphants. Reduced syntheses of free ribosomal subunits and impaired assembly of polysomes are observed in the urb1 mutant as well as in the mtor and raptor morphants, which can be rescued by the Urb1 overexpression. These data demonstrate that Urb1 plays an important role in governing ribosome biogenesis and protein synthesis downstream of mammalian/mechanistic target of rapamycin complex 1(mTORC1), thus regulating the development of digestive organs. Our study indicates the requirement of hyperactive protein synthesis for the digestive organ development.     

Capn8 promoter directs the expression of Cre recombinase in gastric pit cells of transgenic mice

Gastric pit cells are high‐turnover epithelial cells of the gastric mucosa. They secrete mucus to protect the gastric epithelium from acid and pepsin. To investigate the genetic mechanisms underlying the physiological functions of gastric pit cells, we generated a transgenic mouse line, namely, Capn8‐Cre, in which the expression of Cre recombinase was controlled by the promoter of the intracellular Ca²⁺‐regulated cysteine protease calpain‐8. To test the tissue distribution and excision activity of Cre recombinase, the Capn8‐Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4^Co/Co). Multiple‐tissue PCR and LacZ staining demonstrated that Capn8‐Cre transgenic mouse expressed Cre recombinase in the gastric pit cells. Cre recombinase activity was also detected in the liver and skin tissues. These data suggest that the Capn8‐Cre mouse line described here could be used to dissect gene function in gastric pit cells. genesis 47:674–679, 2009. © 2009 Wiley‐Liss, Inc.     

Exosomes from adipose-derived mesenchymal stem cells alleviate liver ischaemia reperfusion injury subsequent to hepatectomy in rats by regulating mitochondrial dynamics and biogenesis

Hepatic ischaemia reperfusion injury (HIRI) is a major factor leading to liver dysfunction after liver resection and liver transplantation. Adipose-derived mesenchymal stem cells (ADSCs) have potential therapeutic effects on HIRI. Exosomes derived from ADSCs (ADSCs-exo) have been widely studied as an alternative of ADSCs therapy. Thus, the aim of this study was to evaluate the potential protective effect and related mechanism of ADSCs-exo on HIRI subsequent to hepatectomy. Rats were randomly divided into four groups: Sham, I30R+PH, ADSCs and ADSCs-exo group. After 24 h of reperfusion, liver and serum of the rats were immediately collected. ADSCs-exo improved liver function, inhibited oxidative stress and reduced apoptosis of hepatocytes in HIRI subsequent to hepatectomy in rats. ADSCs-exo significantly promoted the recovery of mitochondrial function, markedly increased the content of ATP in the liver tissue, and improved the ultrastructure of mitochondria in hepatocytes. Moreover, ADSCs-exo significantly increased the expression of OPA-1, MFN-1 and MFN-2 proteins related to mitochondrial fusion, while DRP-1 and Fis-1 mRNA and protein expression associated with mitochondrial fission were significantly decreased after the treatment with ADSCs-exo. In addition, ADSCs-exo significantly increased the expression of PGC-1α, NRF-1 and TFAM genes and proteins related to mitochondrial biogenesis. ADSCs-exo improves liver function induced by HIRI subsequent to hepatectomy in rats and maintains mitochondrial homeostasis by inhibiting mitochondrial fission, promoting mitochondrial fusion and promoting mitochondrial biogenesis. Therefore, ADSCs-exo may be considered as a potential promising alternative to ADSCs in the treatment of HIRI subsequent to hepatectomy.     

Calpain as a Novel Regulator of Autophagosome Formation

Ubiquitously expressed mu- and m-calpain proteases consist of 80-kDa catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the calpain small 1 (Capns1) gene.

The mu- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. We have found that Capns1 depletion is coupled to increased sensitivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells. Therefore we investigated the involvement of calpains in autophagy using MEFs derived from Capns1 knockout mice and Capns1 depleted human cells as model systems.

We found that autophagy is impaired in Capns1-deficient cells by immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments. Accordingly, the enhancement of lysosomal activity and long-lived proteins degradation, normally occurring upon starvation, are also reduced.

In Capns1-depleted cells ectopic LC3 accumulates in early endosome-like vesicles that might represent a salvage pathway for protein degradation when autophagy is defective.

Addendum to:

Calpain is Required for Macroautophagy in Mammalian Cells

Francesca Demarchi, Cosetta Bertoli, Tamara Copetti, Isei Tanida, Claudio Brancolini, Eeva-Liisa Eskelinen and Claudio Schneider

J Cell Biol 2006; 175:595-605     

Secreted protein with altered thrombospondin repeat (SPATR) is essential for asexual blood stages but not required for hepatocyte invasion by the malaria parasite Plasmodium berghei

Upon entering its mammalian host, the malaria parasite productively invades two distinct cell types, that is, hepatocytes and erythrocytes during which several adhesins/invasins are thought to be involved. Many surface-located proteins containing thrombospondin Type I repeat (TSR) which help establish host–parasite molecular crosstalk have been shown to be essential for mammalian infection. Previous reports indicated that antibodies produced against Plasmodium falciparum secreted protein with altered thrombospondin repeat (SPATR) block hepatocyte invasion by sporozoites but no genetic evidence of its contribution to invasion has been reported. After failing to generate Spatr knockout in Plasmodium berghei blood stages, a conditional mutagenesis system was employed. Here, we show that SPATR plays an essential role during parasite's blood stages. Mutant salivary gland sporozoites exhibit normal motility, hepatocyte invasion, liver stage development and rupture of the parasitophorous vacuole membrane resulting in merosome formation. But these mutant hepatic merozoites failed to establish a blood stage infection in vivo. We provide direct evidence that SPATR is not required for hepatocyte invasion but plays an essential role during the blood stages of P. berghei.     

36 Identification and characterization of small subunit ribosomal intermediates

Bacterial ribosome biogenesis is poorly understood especially in terms of the role of ribosomal RNA (rRNA) maturation in vivo. A major problem in addressing these questions are asynchronous biogenesis, a large population of mature particles and the lack of techniques to isolated in vivo formed ribosome biogenesis intermediates. Our group has taken multiple approaches to allow study of ribosome biogenesis in Escherichia coli. We have used genetic manipulation to discover that for specific biogenesis factors, there is a delicate balance that is necessary for viability. Additionally, we have pioneered an affinity purification approach to allow for isolation of in vivo formed intermediates. Data will be present on our findings for the role of rRNA maturation in biogenesis, subsequent ribosome function, and cell viability. Our findings may result in identification of novel targets for antimicrobial development.     

Loss of c-Met Disrupts Gene Expression Program Required for G2/M Progression during Liver Regeneration in Mice

Background

Previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Met^fl/fl;Alb-Cre^+/− conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration.

Methodology/Principal Finding

The priming events appeared to be intact in Met^fl/fl;Alb-Cre^+/− livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Met^fl/fl;Alb-Cre^+/− primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures.

Conclusion/Significance

In conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.     

m-Calpain is required for preimplantation embryonic development in mice

Background  

μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4 ^-/- mice.     

Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and up-regulates a cohort of ribosome biogenesis genes in zebrafish

Yeast Rcl1 is a potential endonuclease that mediates pre-RNA cleavage at the A₂-site to separate 18S rRNA from 5.8S and 25S rRNAs. However, the biological function of Rcl1 in opisthokonta is poorly defined. Moreover, there is no information regarding the exact positions of 18S pre-rRNA processing in zebrafish. Here, we report that zebrafish pre-rRNA harbours three major cleavage sites in the 5′ETS, namely –477nt (A′-site), –97nt (A₀-site) and the 5′ETS and 18S rRNA link (A₁-site), as well as two major cleavage regions within the ITS1, namely 208–218nt (site 2) and 20–33nt (site E). We also demonstrate that depletion of zebrafish Rcl1 mainly impairs cleavage at the A₁-site. Phenotypically, rcl1^–/– mutants exhibit a small liver and exocrine pancreas and die before 15 days post-fertilization. RNA-seq analysis revealed that the most significant event in rcl1^–/– mutants is the up-regulated expression of a cohort of genes related to ribosome biogenesis and tRNA production. Our data demonstrate that Rcl1 is essential for 18S rRNA maturation at the A₁-site and for digestive organogenesis in zebrafish. Rcl1 deficiency, similar to deficiencies in other ribosome biogenesis factors, might trigger a common mechanism to upregulate the expression of genes responsible for ribosome biogenesis.     

The various impacts of the hepatitis C virus core protein upon hepatic oxidative stress after common bile duct ligation and partial hepatectomy

Abstract

Introduction: Although it is uncertain how the hepatitis C virus (HCV) core protein influences hepatic oxidative stress after partial hepatectomy and common bile duct ligation (CBDL) this may be crucial for the prognosis of patients with HCV infection who have undergone hepatic resection, or who have complications due to a biliary tract obstruction.

Materials and methods: A group of double transgenic mice (DTM) that express both the tetracycline transactivator (tTA) and the HCV core, with conditional, acute expression of the HCV core in the context of the mature liver were subjected to 43% partial hepatectomy and CBDL. The levels of thioredoxin-1, thiobarbituric acid reactive substances (TBARS), and 4-hydroxynonenal (4-HNE) were evaluated in liver samples taken 3 days after the operations.

Results: The DTM had significantly higher TBARS levels than mice that were transgenic for only tTA (i.e. single transgenic mice; STM) and non-transgenic mice (NTM) after a sham laparotomy, CBDL and partial hepatectomy. Of the DTM, the TBARS levels were higher in female mice than in males after a sham laparotomy (P = 0.02) and CBDL (P = 0.0001). 4-HNE staining data were compatible with these results. Furthermore, male DTM exhibited higher levels of thioredoxin-1 than female DTM after sham laparotomy (P = 0.012) and CBDL (P = 0.008).

Conclusions: The HCV core increases hepatic oxidative stress in vivo and female DTM are more vulnerable to the oxidative stress caused by acute core expression with, or without, CBDL. The fact that the female DTM had lower thioredoxin-1 levels may account for this observation.     

RBM19 is essential for preimplantation development in the mouse

Background  

RNA-binding motif protein 19 (RBM19, NCBI Accession # NP_083038) is a conserved nucleolar protein containing 6 conserved RNA recognition motifs. Its biochemical function is to process rRNA for ribosome biogenesis, and it has been shown to play a role in digestive organ development in zebrafish. Here we analyzed the role of RBM19 during mouse embryonic development by generating mice containing a mutation in the Rbm19 locus via gene-trap insertion.     

Role of Wdr45b in maintaining neural autophagy and cognitive function

ABSTRACT

Macroautophagy/autophagy functions as a quality control mechanism by degrading misfolded proteins and damaged organelles and plays an essential role in maintaining neural homeostasis. The phosphoinositide phosphatidylinositol-3-phosphate (PtdIns3P) effector Atg18 is essential for autophagosome formation in yeast. Mammalian cells contain four Atg18 homologs, belonging to two subclasses, WIPI1 (WD repeat domain, phosphoinositide interacting 1), WIPI2 and WDR45B/WIPI3 (WD repeat domain 45B), WDR45/WIPI4. The role of Wdr45b in autophagy and in neural homeostasis, however, remains unknown. Recent human genetic studies have revealed a potential causative role of WDR45B in intellectual disability. Here we demonstrated that mice deficient in Wdr45b exhibit motor deficits and learning and memory defects. Histological analysis reveals that wdr45b knockout (KO) mice exhibit a large number of swollen axons and show cerebellar atrophy. SQSTM1- and ubiquitin-positive aggregates, which are autophagy substrates, accumulate in various brain regions in wdr45b KO mice. Double KO mice, wdr45b and wdr45, die within one day after birth and exhibit more severe autophagy defects than either of the single KO mice, suggesting that these two genes act cooperatively in autophagy. Our studies demonstrated that WDR45B is critical for neural homeostasis in mice. The wdr45b KO mice provide a model to study the pathogenesis of intellectual disability.

Abbreviations: ACSF: artificial cerebrospinal fluid; AMC: aminomethylcoumarin; BPAN: beta-propeller protein-associated neurodegeneration; CALB1: calbindin 1; CNS: central nervous system; DCN: deep cerebellar nuclei; fEPSP: field excitatory postsynaptic potential; IC: internal capsule; ID: intellectual disability; ISH: in situ hybridization; KO: knockout; LTP: long-term potentiation; MBP: myelin basic protein; MGP: medial globus pallidus; PtdIns3P: phosphoinositide phosphatidylinositol-3-phosphate; WDR45B: WD repeat domain 45B; WIPI1: WD repeat domain, phosphoinositide interacting 1; WT: wild type.