Leo W. J. Klomp

Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease

Background & aims Inflammatory bowel disease (IBD) is characterised by chronic intestinal inflammation, resulting from dysregulation of the mucosal immune system and compromised intestinal epithelial barrier function. The bile salt, nuclear farnesoid X receptor (FXR), was recently implicated in intestinal antibacterial defence and barrier function. The aim of this study was to investigate the therapeutic potential of FXR agonists in the treatment of intestinal inflammation in complementary in vivo and in vitro models. Methods Colitis was induced in wild-type (WT) and Fxr-null mice using dextran sodium sulfate, and in WT mice using trinitrobenzenesulfonic acid. Mice were treated with vehicle or the FXR agonist INT-747, and colitis symptoms were assessed daily. Epithelial permeability assays and cytokine expression analysis were conducted in mouse colon and enterocyte-like cells (Caco-2/HT29) treated with medium or INT-747. Inflammatory cytokine secretion was determined by ELISA in various human immune cell types. Results INT-747-treated WT mice are protected from DSS- and TNBS-induced colitis, as shown by significant reduction of body weight loss, epithelial permeability, rectal bleeding, colonic shortening, ulceration, inflammatory cell infiltration and goblet cell loss. Furthermore, Fxr activation in intestines of WT mice and differentiated enterocyte-like cells downregulates expression of key proinflammatory cytokines and preserves epithelial barrier function. INT-747 significantly decreases tumour necrosis factor α secretion in activated human peripheral blood mononuclear cells, purified CD14 monocytes and dendritic cells, as well as in lamina propria mononuclear cells from patients with IBD. Conclusions FXR activation prevents chemically induced intestinal inflammation, with improvement of colitis symptoms, inhibition of epithelial permeability, and reduced goblet cell loss. Furthermore, FXR activation inhibits proinflammatory cytokine production in vivo in the mouse colonic mucosa, and ex vivo in different immune cell populations. The findings provide a rationale to explore FXR agonists as a novel therapeutic strategy for IBD.

Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome

ARC syndrome (OMIM 208085) is an autosomal recessive multisystem disorder characterized by neurogenic arthrogryposis multiplex congenita, renal tubular dysfunction and neonatal cholestasis with bile duct hypoplasia and low gamma glutamyl transpeptidase (gGT) activity. Platelet dysfunction is common. Affected infants do not thrive and usually die in the first year of life. To elucidate the molecular basis of ARC, we mapped the disease to a 7-cM interval on 15q26.1 and then identified germline mutations in the gene VPS33B in 14 kindreds with ARC. VPS33B encodes a homolog of the class C yeast vacuolar protein sorting gene, Vps33, that contains a Sec1-like domain important in the regulation of vesicle-to-target SNARE complex formation and subsequent membrane fusion.

The gene product Murr1 restricts HIV-1 replication in resting CD4 + lymphocytes

Although human immunodeficiency virus-1 (HIV-1) infects quiescent and proliferating CD4+ lymphocytes, the virus replicates poorly in resting T cells. Factors that block viral replication in these cells might help to prolong the asymptomatic phase of HIV infection; however, the molecular mechanisms that control this process are not fully understood. Here we show that Murr1, a gene product known previously for its involvement in copper regulation, inhibits HIV-1 growth in unstimulated CD4+ T cells. This inhibition was mediated in part through its ability to inhibit basal and cytokine-stimulated nuclear factor (NF)-κB activity. Knockdown of Murr1 increased NF-κB activity and decreased IκB-α concentrations by facilitating phospho-IκB-α degradation by the proteasome. Murr1 was detected in CD4+ T cells, and RNA-mediated interference of Murr1 in primary resting CD4+ lymphocytes increased HIV-1 replication. Through its effects on the proteasome, Murr1 acts as a genetic restriction factor that inhibits HIV-1 replication in lymphocytes, which could contribute to the regulation of asymptomatic HIV infection and the progression of AIDS.

Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis.

Aceruloplasminemia is an autosomal recessive disorder characterized by progressive neurodegeneration of the retina and basal ganglia associated with specific inherited mutations in the ceruloplasmin gene. Clinical and pathologic studies in patients with aceruloplasminemia revealed a marked accumulation of iron in affected parenchymal tissues, a finding consistent with early work identifying ceruloplasmin as a ferroxidase and with recent findings showing an essential role for a homologous copper oxidase in iron metabolism in yeast. The presence of neurologic symptoms in aceruloplasminemia is unique among the known inherited and acquired disorders of iron metabolism; recent studies revealed an essential role for astrocyte-specific expression of ceruloplasmin in iron metabolism and neuronal survival in the central nervous system. Recognition of aceruloplasminemia provides new insights into the genetic and environmental determinants of copper metabolism and has important implications for our understanding of the role of copper in human neurodegenerative diseases.

Characterization of mutations in ATP8B1 associated with hereditary cholestasis

Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are clinically distinct hereditary disorders. PFIC patients suffer from chronic cholestasis and develop liver fibrosis. BRIC patients experience intermittent attacks of cholestasis that resolve spontaneously. Mutations in ATP8B1 (previously FIC1) may result in PFIC or BRIC. We report the genomic organization of ATP8B1 and mutation analyses of 180 families with PFIC or BRIC that identified 54 distinct disease mutations, including 10 mutations predicted to disrupt splicing, 6 nonsense mutations, 11 small insertion or deletion mutations predicted to induce frameshifts, 1 large genomic deletion, 2 small inframe deletions, and 24 missense mutations. Most mutations are rare, occurring in 1–3 families, or are limited to specific populations. Many patients are compound heterozygous for 2 mutations. Mutation type or location correlates overall with clinical severity: missense mutations are more common in BRIC (58% vs. 38% in PFIC), while nonsense, frameshifting, and large deletion mutations are more common in PFIC (41% vs. 16% in BRIC). Some mutations, however, lead to a wide range of phenotypes, from PFIC to BRIC or even no clinical disease. ATP8B1 mutations were detected in 30% and 41%, respectively, of the PFIC and BRIC patients screened. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html) and at www.atp8b1‐primers.nl (HEPATOLOGY 2004;40:27–38.)

A novel role for XIAP in copper homeostasis through regulation of MURR1

XIAP is a potent suppressor of apoptosis that directly inhibits specific members of the caspase family of cysteine proteases. Here we demonstrate a novel role for XIAP in the control of intracellular copper levels. XIAP was found to interact with MURR1, a factor recently implicated in copper homeostasis. XIAP binds to MURR1 in a manner that is distinct from that utilized by XIAP to bind caspases, and consistent with this, MURR1 did not affect the antiapoptotic properties of XIAP. However, cells and tissues derived from Xiap‐deficient mice were found to contain reduced copper levels, while suppression of MURR1 resulted in increased intracellular copper in cultured cells. Consistent with these opposing effects, XIAP was observed to negatively regulate MURR1 protein levels by the formation of K48 polyubiquitin chains on MURR1 that promote its degradation. These findings represent the first described phenotypic alteration in Xiap‐deficient mice and demonstrate that XIAP can function through MURR1 to regulate copper homeostasis.

Biochemical characterization and subcellular localization of human copper transporter 1 (hCTR1).

The human copper transporter 1 gene (hCTR1) was previously identified by functional complementation in ctr1-deficient yeast. Overexpression of hCTR1 in wild-type yeast leads to increased sensitivity to copper toxicity, and mice with a homozygous disruption at the Ctr1 locus die early during embryogenesis. It is proposed that hCTR1 is responsible for high-affinity copper uptake into human cells, but the underlying molecular mechanisms are unknown. To begin to investigate the biochemical characteristics of hCTR1, a polyclonal antiserum was raised against recombinant hCTR1-fusion peptides. Biosynthetic studies using this antiserum revealed that hCTR1 was synthesized as a precursor protein of 28 kDa containing N-linked oligosaccharides, and is then converted to a mature protein of approx. 35 kDa, which is ubiquitously expressed. Immunofluorescence studies showed that subcellular hCTR1 localization differed markedly between cell types. In some cell lines, hCTR1 was located predominantly in an intracellular vesicular perinuclear compartment, and in others hCTR1 was located predominantly at the plasma membrane. In contrast with the copper export P-type ATPases mutated in Wilson disease and Menkes disease, the localization of hCTR1 was not influenced by copper concentrations. Inhibition of endocytosis by methyl-beta-cyclodextrin caused a partial redistribution of hCTR1 to the cell surface of HeLa cells. Taken together, the results in this study suggest a cell-specific control of copper uptake, which involves subcellular localization of the hCTR1 protein.

Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake

High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2-vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2-vsvG. Furthermore, hCTR2-vsvG was co-immunoprecipitated with hCTR2-FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2-eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)-luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE-luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 muM CuCl2, consistent with the K(m) of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE-luciferase reporter activation in a copper-dependent manner at 40 and 100 microM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.

Progressive familial intrahepatic cholestasis type 1 and extrahepatic features: no catch-up of stature growth, exacerbation of diarrhea, and appearance of liver steatosis after liver transplantation

BACKGROUND/AIMS Progressive familial intrahepatic cholestasis characterized by normal serum gamma-glutamyltransferase activity can be due to mutations in familial intrahepatic cholestasis type 1 (FIC1) (ATP8B1), a gene expressed in several organs. In some cases, it is associated with extrahepatic features. We searched for FIC1 mutations and analyzed the outcome of extrahepatic features after liver transplantation in two children with this form of progressive familial intrahepatic cholestasis associated with chronic unexplained diarrhea and short stature. METHODS FIC1 sequence was determined after polymerase chain reaction (PCR) of genomic lymphocyte DNA and/or reverse transcription-PCR of liver or lymphocyte RNA. RESULTS A homozygous amino acid change deletion was found in one child. The second child harboured compound heterozygous missense and nonsense mutations. In both children, despite successful liver transplantation, evolution (follow-up: 9.5-11 years) was characterized by exacerbation of diarrhea and no catch-up of stature growth, and appearance of liver steatosis. CONCLUSIONS Progressive familial intrahepatic cholestasis characterized by normal serum gamma-glutamyltransferase activity and extrahepatic features corresponds to progressive familial intrahepatic cholestasis type 1. Extrahepatic symptomatology is not corrected or may be aggravated by liver transplantation, impairing life quality.

The ubiquitously expressed MURR1 protein is absent in canine copper toxicosis.

BACKGROUND/AIMS Copper toxicosis (CT) in Bedlington terriers is an autosomal recessive disorder characterized by massive lysosomal copper accumulation in livers of affected dogs, and a defect in the biliary excretion of this metal. We propose that MURR1, the gene defective in canine CT, has a role in the regulation of copper excretion into bile during copper overload. METHODS Polyclonal antibodies raised against full-length recombinant human MURR1 were used for immunoblot analysis and indirect immunofluorescence studies. RESULTS Using Western blot analysis, these antibodies abundantly detected MURR1 as a 23 kDa protein in liver extracts of mice and dogs, but MURR1 was undetectable in the livers of affected Bedlington terriers. MURR1 was also detected in different tissues and cell lines; in cell lines the protein was found both in cytosol and membrane preparations. Consistent with this observation, indirect immunofluorescence staining revealed that in some cells MURR1 was associated with a vesicular compartment diffusely localized throughout the cell. CONCLUSIONS The genomic deletion in MURR1 results in complete absence of MURR1 protein. Based on the unanticipated subcellular localization, our results suggest a role for MURR1 in the regulation of vesicular copper sequestration during copper overload.