Acetylcholine induces fibrogenic effects via M2/M3 acetylcholine receptors in non-alcoholic steatohepatitis and in primary human hepatic stellate cells.

BACKGROUND: The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear. AIMS: We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH). METHODS: Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis. RESULTS: We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers. CONCLUSION: We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.

Amphiregulin activates human hepatic stellate cells and is upregulated in non alcoholic steatohepatitis.

Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways, and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD.

Sympathetic nervous system catecholamines and neuropeptide Y neurotransmitters are upregulated in human NAFLD and modulate the fibrogenic function of hepatic stellate cells.

BACKGROUND: Sympathetic nervous system (SNS) signalling regulates murine hepatic fibrogenesis through effects on hepatic stellate cells (HSC), and obesity-related hypertension with SNS activation accelerates progression of non-alcoholic fatty liver disease (NAFLD), the commonest cause of chronic liver disease. NAFLD may lead to cirrhosis. The effects of the SNS neurotransmitters norepinephrine (NE), epinephrine (EPI) and neuropeptide Y (NPY) on human primary HSC (hHSC) function and in NAFLD pathogenesis are poorly understood. AIMS: to determine the mechanistic effects of NE/EPI/NPY on phenotypic changes in cultured hHSC, and to study SNS signalling in human NAFLD livers. METHODS: Freshly isolated hHSC were assessed for expression of cathecholamine/neuropeptide Y receptors and for the synthesis of NE/EPI. The effects of NE/EPI/NPY and adrenoceptor antagonists prazosin (PRZ)/propranolol (PRL) on hHSC fibrogenic functions and the involved kinases and interleukin pathways were examined. Human livers with proven NAFLD were then assessed for upregulation of SNS signalling components. RESULTS: Activated hHSC express functional α/ß-adrenoceptors and NPY receptors, which are upregulated in the livers of patients with cirrhotic NAFLD. hHSC in culture synthesize and release NE/EPI, required for their optimal basal growth and survival. Exogenous NE/EPI and NPY dose-dependently induced hHSC proliferation, mediated via p38 MAP, PI3K and MEK signalling. NE and EPI but not NPY increased expression of collagen-1α2 via TGF-ß without involvement of the pro-fibrogenic cytokines leptin, IL-4 and IL-13 or the anti-fibrotic cytokine IL-10. CONCLUSIONS: hHSC synthesize and require cathecholamines for optimal survival and fibrogenic functionality. Activated hHSC express directly fibrogenic α/ß-adrenoceptors and NPY receptors, upregulated in human cirrhotic NAFLD. Adrenoceptor and NPY antagonists may be novel anti-fibrotic agents in human NAFLD.

Maternal obesity during pregnancy and lactation programs the development of offspring non-alcoholic fatty liver disease in mice.

BACKGROUND & AIMS: Obesity induced, non-alcoholic fatty liver disease (NAFLD), is now the major cause in affluent countries, of the spectrum of steatosis-to-cirrhosis. Obesity and NAFLD rates in reproductive age women, and adolescents, are rising worldwide. Our hypothesis was that maternal obesity and lactation transmit to the offspring a pre-disposition to dysmetabolism, obesity and NAFLD. METHODS: Female mice were fed standard or obesogenic chow, before, throughout pregnancy, and during lactation. The critical developmental period was studied by cross-fostering offspring of lean and obese dams. Offspring were then weaned onto standard chow and studied at 3months. Read-outs included markers of metabolic dysfunction, biochemical and histological indicators of NAFLD, induction of liver fibrogenesis, and activation of pro-fibrotic pathways. Mechanisms involved in programming a dysmetabolic and NAFLD phenotype were investigated by assaying breast milk components. RESULTS: Offspring of obese dams had a dysmetabolic, insulin resistant and NAFLD phenotype compared to offspring of lean dams. Offspring of lean dams that were suckled by obese dams showed an exaggerated dysmetabolic and NAFLD phenotype, with increased body weight, as well as increased levels of insulin, leptin, aspartate transaminase, interleukin-6, tumour necrosis factor-alpha, liver triglycerides, steatosis, hepatic fibrogenesis, renal norepinephrine, and liver alpha1-D plus beta1-adrenoceptors, indicative of sympathetic nervous system activation. Obese dams also had raised breast milk leptin levels compared to lean dams. CONCLUSIONS: Maternal obesity programs development of a dysmetabolic and NAFLD phenotype, which is critically dependent on the early postnatal period and possibly involving alteration of hypothalamic appetite nuclei signalling by maternal breast milk and neonatal adipose tissue derived, leptin.

Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex.

TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are found together as components of several highly conserved chromatin-modifying complexes. Both proteins show sequence homology to bacterial RuvB but the nature and mechanism of their biochemical role remain unknown. Recombinant human TIP48 and TIP49 were assembled into a stable high molecular mass equimolar complex and tested for activity in vitro. TIP48/TIP49 complex formation resulted in synergistic increase in ATPase activity but ATP hydrolysis was not stimulated in the presence of single-stranded, double-stranded or four-way junction DNA and no DNA helicase or branch migration activity could be detected. Complexes with catalytic defects in either TIP48 or TIP49 had no ATPase activity showing that both proteins within the TIP48/TIP49 complex are required for ATP hydrolysis. The structure of the TIP48/TIP49 complex was examined by negative stain electron microscopy. Three-dimensional reconstruction at 20 A resolution revealed that the TIP48/TIP49 complex consisted of two stacked hexameric rings with C6 symmetry. The top and bottom rings showed substantial structural differences. Interestingly, TIP48 formed oligomers in the presence of adenine nucleotides, whilst TIP49 did not. The results point to biochemical differences between TIP48 and TIP49, which may explain the structural differences between the two hexameric rings and could be significant for specialised functions that the proteins perform individually.

Relocalization of human chromatin remodeling cofactor TIP48 in mitosis.

TIP48 is a highly conserved eukaryotic AAA+ protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis, TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis.

The role of RuvA octamerization for RuvAB function in vitro and in vivo.

RuvA plays an essential role in branch migration of the Holliday junction by RuvAB as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli. Two types of RuvA-Holliday junction complexes have been characterized: 1) complex I containing a single RuvA tetramer and 2) complex II in which the junction is sandwiched between two RuvA tetramers. The functional differences between the two forms are still not clear. To investigate the role of RuvA octamerization, we introduced three amino acid substitutions designed to disrupt the E. coli RuvA tetramer-tetramer interface as identified by structural studies. The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nm. We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions. The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions. However, junction binding and the efficiency of branch migration of four-way junctions were affected. The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer. Significantly, the mutant failed to complement the UV sensitivity of E. coli DeltaruvA cells. These results indicate strongly that RuvA octamerization is essential for the full biological activity of RuvABC.

Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance.

The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant structural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the alpha-helices that form this bundle show homology to a putative DNA-binding motif known as SAP. To investigate the biochemical function of the triple-helix domain, truncated Ydc2 mutants were expressed in E. coli and in fission yeast. Although the truncated proteins retained all amino-acid residues that map to the structural core of RuvC including the catalytic site, deletion of the SAP motif alone or the whole triple-helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to synthetic junctions in vitro. These results are in full agreement with our proposal for a DNA-binding role of the triple-helix motif [Ceschini et al. (2001) EMBO J. 20, 6601-6611]. The biological effect of Ydc2 on mtDNA in yeast was probed using wild-type and several Ydc2 mutants expressed in Deltaydc2 S. pombe. The truncated mutants were shown to localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. Plasmids expressing full-length Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA 'phenotype'. These results provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pombe.