Nuclear Receptor Subfamily 4A Signaling as a Key Disease Pathway of CD1c+ Dendritic Cell Dysregulation in Systemic Sclerosis.

OBJECTIVE: This study was undertaken to identify key disease pathways driving conventional dendritic cell (cDC) alterations in systemic sclerosis (SSc). METHODS: Transcriptomic profiling was performed on peripheral blood CD1c+ cDCs (cDC2s) isolated from 12 healthy donors and 48 patients with SSc, including all major disease subtypes. We performed differential expression analysis for the different SSc subtypes and healthy donors to uncover genes dysregulated in SSc. To identify biologically relevant pathways, we built a gene coexpression network using weighted gene correlation network analysis. We validated the role of key transcriptional regulators using chromatin immunoprecipitation (ChIP) sequencing and in vitro functional assays. RESULTS: We identified 17 modules of coexpressed genes in cDCs that correlated with SSc subtypes and key clinical traits, including autoantibodies, skin score, and occurrence of interstitial lung disease. A module of immunoregulatory genes was markedly down-regulated in patients with the diffuse SSc subtype characterized by severe fibrosis. Transcriptional regulatory network analysis performed on this module predicted nuclear receptor 4A (NR4A) subfamily genes (NR4A1, NR4A2, NR4A3) as the key transcriptional regulators of inflammation. Indeed, ChIP-sequencing analysis indicated that these NR4A members target numerous differentially expressed genes in SSc cDC2s. Inclusion of NR4A receptor agonists in culture-based experiments provided functional proof that dysregulation of NR4As affects cytokine production by cDC2s and modulates downstream T cell activation. CONCLUSION: NR4A1, NR4A2, and NR4A3 are important regulators of immunosuppressive and fibrosis-associated pathways in SSc cDCs. Thus, the NR4A family represents novel potential targets to restore cDC homeostasis in SSc.

CXCL4 drives fibrosis by promoting several key cellular and molecular processes.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

Hypoxia and TLR9 activation drive CXCL4 production in systemic sclerosis plasmacytoid dendritic cells via mtROS and HIF-2α.

OBJECTIVE: SSc is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified chemokine (C-X-C motif) ligand 4 (CXCL4) as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production. METHODS: Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several toll-like receptor (TLR) agonists. Further, pro-inflammatory cytokine production, CXCL4, hypoxia-inducible factor (HIF) -1α and HIF-2α gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays. RESULTS: CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (P < 0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (P = 0.0079) leading to stabilization of HIF-2α (P = 0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord CD34 derived pDCs. CONCLUSION: TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2α pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4.

Transcriptome Analysis of CCR9+ T Helper Cells From Primary Sjögren's Syndrome Patients Identifies CCL5 as a Novel Effector Molecule.

Introduction: CCR9+ Tfh-like pathogenic T helper (Th) cells are elevated in patients with primary Sjögren's syndrome (pSS) and indicated to play a role in pSS immunopathology. Here we delineate the CCR9+ Th cell-specific transcriptome to study the molecular dysregulation of these cells in pSS patients. Methods: CCR9+, CXCR5+ and CCR9-CXCR5- Th cells from blood of 7 healthy controls (HC) and 7 pSS patients were FACS sorted and RNA sequencing was performed. Computational analysis was used to identify differentially expressed genes (DEGs), coherent gene expression networks and differentially regulated pathways. Target genes were replicated in additional cohorts. Results: 5131 genes were differentially expressed between CCR9+ and CXCR5+ Th cells; 6493 and 4783 between CCR9+ and CCR9-CXCR5- and between CXCR5+ and CCR9-CXCR5-, respectively. In the CCR9+ Th cell subset 2777 DEGs were identified between HC and pSS patients, 1416 and 1077 in the CXCR5+ and CCR9-CXCR5- subsets, respectively. One gene network was selected based on eigengene expression differences between the Th cell subsets and pathways enriched for genes involved in migration and adhesion, cytokine and chemokine production. Selected DEGs of interest (HOPX, SOX4, ITGAE, ITGA1, NCR3, ABCB1, C3AR1, NT5E, CCR5 and CCL5) from this module were validated and found upregulated in blood CCR9+ Th cells, but were similarly expressed in HC and pSS patients. Increased frequencies of CCR9+ Th cells were shown to express higher levels of CCL5 than CXCR5+ and CCR9-CXCR5- Th cells, with the highest expression confined to effector CCR9+ Th cells. Antigenic triggering and stimulation with IL-7 of the Th cell subsets co-cultured with monocytes strongly induced CCL5 secretion in CCR9+ Th cell cocultures. Additionally, effector CCR9+ Th cells rapidly released CCL5 and secreted the highest CCL5 levels upon stimulation. Conclusion: Transcriptomic analysis of circulating CCR9+ Th cells reveals CCR9-specific pathways involved in effector T cell function equally expressed in pSS patients and HC. Given the increased numbers of CCR9+ Th cells in the blood and inflamed glands of pSS patients and presence of inflammatory stimuli to activate these cells this suggests that CCR9-specific functions, such as cell recruitment upon CCL5 secretion, could significantly contribute to immunopathology in pSS.

Characterization of Long Non-Coding RNAs in Systemic Sclerosis Monocytes: A Potential Role for PSMB8-AS1 in Altered Cytokine Secretion.

Systemic sclerosis (SSc) is a chronic autoimmune disease mainly affecting the connective tissue. In SSc patients, monocytes are increased in circulation, infiltrate affected tissues, and show a pro-inflammatory activation status, including the so-called interferon (IFN) signature. We previously demonstrated that the dysregulation of the IFN response in SSc monocytes is sustained by altered epigenetic factors as well as by upregulation of the long non-coding RNA (lncRNA) NRIR. Considering the enormously diverse molecular functions of lncRNAs in immune regulation, the present study investigated the genome-wide profile of lncRNAs in SSc monocytes, with the aim to further unravel their possible role in monocyte dysregulation and disease pathogenesis. Transcriptomic data from two independent cohorts of SSc patients identified 886 lncRNAs with an altered expression in SSc monocytes. Differentially expressed lncRNAs were correlated with neighboring protein coding genes implicated in the regulation of IFN responses and apoptotic signaling in SSc monocytes. In parallel, gene co-expression network analysis identified the lncRNA PSMB8-AS1 as a top-ranking hub gene in co-expression modules implicated in cell activation and response to viral and external stimuli. Functional characterization of PSMB8-AS1 in monocytes demonstrated that this lncRNA is involved in the secretion of IL-6 and TNFα, two pivotal pro-inflammatory cytokines altered in the circulation of SSc patients and associated with fibrosis and disease severity. Collectively, our data showed that lncRNAs are linked to monocyte dysregulation in SSc, and highlight their potential contribution to disease pathogenesis.

Implication of miR-126 and miR-139-5p in Plasmacytoid Dendritic Cell Dysregulation in Systemic Sclerosis.

Compelling evidence shows the involvement of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc) pathogenesis. This study investigated whether microRNAs (miRNAs) are involved in the dysregulation of pDCs in SSc patients already at early stages. RNA from circulating pDCs was isolated from two independent cohorts of SSc patients with different disease phenotypes, and individuals with Raynaud's phenomenon, for microRNA profiling and RNA-sequencing analysis. Proteomic analysis was exploited to identify novel direct miRNA targets at the protein level. Twelve and fifteen miRNAs were differentially expressed in at least one group of patients compared to healthy controls in discovery cohort I and II, respectively. Of note, miR-126 and miR-139-5p were upregulated in both preclinical and definite SSc patients and correlated with the expression of type I interferon (IFN)-responsive genes. Toll-like receptor 9 (TLR9) stimulation of healthy pDCs upregulated the expression of both miRNAs, similarly to what was observed in patients. The proteomic analysis identified USP24 as a novel target of miR-139-5p. The expression level of USP24 was inversely correlated with miR-139-5p expression in SSc patients and induced by TLR9 stimulation in healthy pDCs. These findings demonstrated that the miRNA profile is altered in pDCs of SSc patients already at early stages of the disease and indicate their potential contribution to pDC activation observed in patients.

CXCL4 triggers monocytes and macrophages to produce PDGF-BB, culminating in fibroblast activation: Implications for systemic sclerosis.

OBJECTIVE: To analyze how monocyte and macrophage exposure to CXCL4 induces inflammatory and fibrotic processes observed in Systemic sclerosis (SSc) patients. METHODS: In six independent experiments, monocytes of healthy controls (HC) and SSc patients were stimulated with CXCL4, TLR-ligands, IFNɑ or TGFß and the secretion of cytokines in the supernatant was assessed by multiplex immunoassays. PDGF-BB production by monocyte-derived macrophages was quantified using immunoassays. The number of monocytes and PDGF-BB in circulation was quantified in HC and SSc patients with the Sysmex XT-1800i haematology counter and immunoassays. Intracellular PDGF-BB was quantified in monocytes by Western blot. PDGF-receptor inhibition was achieved using siRNA-mediated knockdown or treatment with Crenolanib. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblasts was analyzed by qPCR, ELISA and ECM deposition assays. RESULTS: SSc and HC monocytes released PDGF-BB upon stimulation with CXCL4. Conversely, TLR ligands, IFNɑ or TGFß did not induce PDGF-bb release. PDGF-BB plasma levels were significantly (P = 0.009) higher in diffuse SSc patients (n = 19), compared with HC (n = 21). In healthy dermal fibroblasts, PDGF-BB enhanced TNFɑ-induced expression of inflammatory cytokines and increased ECM production. Comparable results were observed in fibroblasts cultured in supernatant taken from macrophages stimulated with CXCL4. This effect was almost completely abrogated by inhibition of the PDGF-receptor using Crenolanib. CONCLUSION: Our findings demonstrate that CXCL4 can drive fibroblast activation indirectly via PDGF-BB production by myeloid cells. Hence, targeting PDGF-BB or CXCL4-induced PDGF-BB release could be clinically beneficial for patients with SSc.

mTOR inhibition by metformin impacts monosodium urate crystal-induced inflammation and cell death in gout: a prelude to a new add-on therapy?

OBJECTIVE: Gout is the most common inflammatory arthritis worldwide, and patients experience a heavy burden of cardiovascular and metabolic diseases. The inflammation is caused by the deposition of monosodium urate (MSU) crystals in tissues, especially in the joints, triggering immune cells to mount an inflammatory reaction. Recently, it was shown that MSU crystals can induce mechanistic target of rapamycin (mTOR) signalling in monocytes encountering these crystals in vitro. The mTOR pathway is strongly implicated in cardiovascular and metabolic disease. We hypothesised that inhibiting this pathway in gout might be a novel avenue of treatment in these patients, targeting both inflammation and comorbidities. METHODS: We used a translational approach starting from ex vivo to in vitro and back to in vivo. RESULTS: We show that ex vivo immune cells from patients with gout exhibit higher expression of the mTOR pathway, which we can mimic in vitro by stimulating healthy immune cells (B lymphocytes, monocytes, T lymphocytes) with MSU crystals. Monocytes are the most prominent mTOR expressers. By using live imaging, we demonstrate that monocytes, on encountering MSU crystals, initiate cell death and release a wide array of proinflammatory cytokines. By inhibiting mTOR signalling with metformin or rapamycin, a reduction of cell death and release of inflammatory mediators was observed. Consistent with this, we show that patients with gout who are treated with the mTOR inhibitor metformin have a lower frequency of gout attacks. CONCLUSIONS: We propose mTOR inhibition as a novel therapeutic target of interest in gout treatment.

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting.

BACKGROUND AND OBJECTIVE: Systemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. METHODS: Chromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression. RESULTS: 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. CONCLUSION: SSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.

Galectin-9 is an easy to measure biomarker for the interferon signature in systemic lupus erythematosus and antiphospholipid syndrome.

OBJECTIVE: The interferon (IFN) signature is related to disease activity and vascular disease in systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and represents a promising therapeutic target. Quantification of the IFN signature is currently performed by gene expression analysis, limiting its current applicability in clinical practice. Therefore, the objective of this study was to establish an easy to measure biomarker for the IFN signature. METHODS: Serum levels of galectin-9, CXCL-10 (IP-10) and tumour necrosis factor receptor type II (TNF-RII) were measured in patients with SLE, SLE+APS and primary APS (PAPS) and healthy controls (n=148) after an initial screening of serum analytes in a smaller cohort (n=43). Analytes were correlated to measures of disease activity and the IFN signature. The performance of galectin-9, CXCL-10 and TNF-RII as biomarkers to detect the IFN signature was assessed by receiver operating characteristic curves. RESULTS: Galectin-9, CXCL-10 and TNF-RII were elevated in patients with SLE, SLE+APS and PAPS (p<0.05) and correlated with disease activity and tissue factor expression. Galectin-9 correlated stronger than CXCL-10 or TNF-RII with the IFN score (r=0.70, p<0.001) and was superior to CXCL-10 or TNF-RII in detecting the IFN signature (area under the curve (AUC) 0.86). Importantly, in patients with SLE(±APS), galectin-9 was also superior to anti-dsDNA antibody (AUC 0.70), or complement C3 (AUC 0.70) and C4 (AUC 0.78) levels in detecting the IFN signature. CONCLUSION: Galectin-9 is a novel, easy to measure hence clinically applicable biomarker to detect the IFN signature in patients with systemic autoimmune diseases such as SLE and APS.

Serum microRNA screening and functional studies reveal miR-483-5p as a potential driver of fibrosis in systemic sclerosis.

OBJECTIVE: MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease. METHODS: The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (n = 22), systemic lupus erythematosus (SLE) (n = 33) and primary Sjögren's syndrome (pSS) (n = 23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells. RESULTS: 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes. CONCLUSIONS: Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc.

Telomere quantification in frontal and temporal brain tissue of patients with schizophrenia.

Recent imaging studies have suggested that accelerated aging occurs in schizophrenia. However, the exact cause of these findings is still unclear. In this study we measured telomere length, a marker for cell senescence, in gray and white matter brain tissue from the medial frontal gyrus (MFG) and superior temporal gyrus (STG) of 9 patients with schizophrenia and 11 controls. No alterations in telomere length were found in MFG gray and white matter and in STG gray matter. A significant reduction in telomere length was observed in STG white matter of patients with schizophrenia as compared to controls (fold change of -0.42, U = 5, P = 0.008). Our results support previous findings that telomere length in gray matter is not affected, whereas they suggest that increased cell senescence may affect white matter temporal brain tissue.

Association of MicroRNA-618 Expression With Altered Frequency and Activation of Plasmacytoid Dendritic Cells in Patients With Systemic Sclerosis.

OBJECTIVE: Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFNα than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc. METHODS: We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. RESULTS: Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFNα, mimicking the PDC phenotype observed in SSc patients. CONCLUSION: Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFNα, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.

Rescue of defective ATP8B1 trafficking by CFTR correctors as a therapeutic strategy for familial intrahepatic cholestasis.

BACKGROUND & AIMS: ATP8B1 deficiency is an autosomal recessive liver disease characterized by intrahepatic cholestasis. ATP8B1 mutation p.I661T, the most frequent mutation in European patients, results in protein misfolding and impaired targeting to the plasma membrane. Similarly, mutations in cystic fibrosis transmembrane conductance regulator (CFTR), associated with cystic fibrosis, impair protein folding and trafficking. The aim of this study was to investigate whether compounds that rescue CFTR F508del trafficking are capable of improving p.I661T-ATP8B1 plasma membrane expression. METHODS: The effect of CFTR corrector compounds on plasma membrane expression of p.I661T-ATP8B1 was evaluated by cell surface biotinylation and immunofluorescence. ATPase activity was evaluated of a purified analogue protein carrying a mutation at the matching position (p.L622T-ATP8A2). RESULTS: The clinically used compounds, 4-phenylbutyric acid (4-PBA), suberoylanilide hydroxamic acid (SAHA) and N-butyldeoxynojirimycin (NB-DNJ) improved p.I661T-ATP8B1 plasma membrane targeting. Compounds C4, C5, C13 and C17 also significantly increased plasma membrane expression of p.I661T-ATP8B1. SAHA and compound C17 upregulated ATP8B1 transcription. p.I661T-ATP8B1 was partly targeted to the canalicular membrane in polarized cells, which became more evident upon treatment with SAHA and/or C4. p.L622T-ATP8A2 showed phospholipid-induced ATPase activity, suggesting that mutations at a matching position in ATP8B1 do not block functionality. Combination therapy of SAHA and compound C4 resulted in an additional improvement of ATP8B1 cell surface abundance. CONCLUSIONS: This study shows that several CFTR correctors can improve trafficking of p.I661T-ATP8B1 to the plasma membrane in vitro. Hence, these compounds may be suitable to be part of a future therapy for ATP8B1 deficiency and other genetic disorders associated with protein misfolding. LAY SUMMARY: Compounds that improve the cellular machinery dealing with protein homeostasis (proteostasis) and allow for proper folding of proteins with (mild) missense mutations are called proteostasis regulators (Balch, Science 2008). Such compounds are potentially of high therapeutic value for many (liver) diseases. In this manuscript, we investigated whether compounds identified in screens as CFTR folding correctors are actually proteostasis regulators and thus have a broader application in other protein folding diseases. Using these compounds, we could indeed show improved trafficking to the (apical) plasma membrane of a mutated ATP8B1 protein, carrying the p.I661T missense mutation. This is the most frequently identified mutation in this rare cholestatic disorder. Importantly, ATP8B1 shows no similarity to CFTR. These data are important in providing support for the concept that rare, genetic liver diseases can potentially be treated using a generalized strategy.

Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na(+) -taurocholate cotransporting polypeptide knockout mice.

UNLABELLED: The Na(+) -taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte-specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1(-/-) hepatocytes showed absence of sodium-dependent taurocholic acid uptake, whereas sodium-independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1(-/-) mice, explained by increased Asbt and Ostα/ß expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down-regulation of OATP1A1 and up-regulation of OATP1A4. Furthermore, sodium-dependent taurocholic acid uptake was inhibited by Myrcludex B in wild-type hepatocytes, while Slc10a1(-/-) hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1(-/-) mice. CONCLUSION: The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation.

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression.

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P(1B)-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.

Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation.

Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1(Δhep)) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1(Δhep) mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1(Δhep) mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1(Δhep) mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1(Δhep) mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1(Δhep) mice could be detected. Despite the absence of hepatocellular toxicity in Commd1(Δhep) mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.

Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P4-ATPases.

Members of the P(4) family of P-type ATPases (P(4)-ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P(4)-ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P(4)-ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P(4)-ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P(4)-ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P(4)-ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed.