Epitope-specific anti-nuclear antibodies are expressed in a mouse model of primary biliary cirrhosis and are cytokine-dependent.

Although the hallmark of primary biliary cirrhosis (PBC) is the presence of anti-mitochondrial antibodies (AMA), a significant number of patients have anti-nuclear antibodies (ANA) directed primarily against two nuclear proteins, gp210 and sp100. In PBC, there are considerable data on the specificity of these anti-nuclear antibodies as well as suggestive evidence that antibodies to gp210 predict a poor outcome. However, a further understanding of the significance of these autoantibodies has been hampered by limitations in accessing human subjects in a preclinical or early asymptomatic stage. To overcome this limitation, we have taken advantage of transgenic mice with abrogated transforming growth factor-ß signalling in T cells (dnTGF-ßRII) that develop histological features of PBC as well as the same AMA specificity. We studied these mice for serum ANA, including specific autoantibodies against gp210 and sp100. We further examined sera from dnTGF-ßRII mice with concurrent deletions of the genes encoding interleukin (IL)-12p35, IL-12p40, IL-23p19, IL-17, IL-6, interferon (IFN)-γ or tumour necrosis factor (TNF)-α. Sera from all the dnTGF-ßRII mouse lines contained antibodies against gp210 and sp100. Of significance, mice with germline deletions of the genes encoding IL-12p40, IL-23p19, IL-17, IL-6 and TNF-α had significantly lower titres of anti-gp210 antibodies. These results provide a platform to dissect the mechanisms of gp210 and sp100 autoantibody production in dnTGF-ßRII mice as well as to study the possible role of ANA in the pathophysiology of PBC.

Autoantibodies from patients with primary biliary cirrhosis recognize a restricted region within the cytoplasmic tail of nuclear pore membrane glycoprotein Gp210.

Patients with primary biliary cirrhosis (PBC) frequently have autoantibodies against a 210-kD integral glycoprotein of the nuclear envelope pore membrane. This protein, termed gp210, has a 1,783-amino acid amino-terminal domain located in the perinuclear space, a 20-amino acid transmembrane segment, and a 58-amino acid cytoplasmic carboxy-terminal tail. We now demonstrate that autoantibodies from 25 patients with PBC that recognize gp210 react with the cytoplasmic carboxy-terminal tail while none react with unmodified linear epitopes in the amino-terminal domain. The epitope(s) recognized by autoantibodies from all 25 patients is contained within a stretch of 15 amino acids. The recognized amino acid sequence is homologous to the protein products of the Escherichia coli mutY gene and Salmonella typhimurium mutB gene with an exact identity of six consecutive amino acids, suggesting that anti-gp210 antibodies may arise by molecular mimicry of bacterial antigenic determinants.

Identification and characterization of autoantibodies against the nuclear envelope lamin B receptor from patients with primary biliary cirrhosis.

We have identified autoantibodies from two patients with primary biliary cirrhosis (PBC) that recognize the nuclear envelope of mammalian cells on indirect immunofluorescence microscopy. These antibodies bind to a 58-kD integral membrane protein (p58) of the turkey erythrocyte nuclear envelope, which has been previously identified as a membrane receptor for lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531). The antibodies also bind to a 61-kD integral membrane protein (p61) of the rat liver nuclear envelope. Affinity-purified antibodies eluted from turkey p58 bind to rat p61, showing that the two proteins share an epitope(s) and that p61 is likely the rat liver lamin B receptor. In human nuclear envelopes, the antigen recognized has an apparent molecular mass close to that of avian protein. These findings, along with the previous discovery of autoantibodies against an integral membrane glycoprotein (gp210) of the nuclear pore membrane in patients with PBC, suggest that antibodies against integral membrane proteins of the nuclear envelope are characteristic of a subset of patients with PBC.

The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal.

The lamin B receptor (LBR) is a polytopic protein of the inner nuclear membrane. It is synthesized without a cleavable amino-terminal signal sequence and composed of a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. To identify a nuclear envelope targeting signal, we have examined the cellular localization by immunofluorescence microscopy of chicken LBR, its amino-terminal domain and chimeric proteins transiently expressed in transfected COS-7. Full-length LBR was targeted to the nuclear envelope. The amino-terminal domain, without any transmembrane segments, was transported to the nucleus but excluded from the nucleolus. When the amino-terminal domain of LBR was fused to the amino-terminal side of a transmembrane segment of a type II integral membrane protein of the ER/plasma membrane, the chimeric protein was targeted to the nuclear envelope, likely the inner nuclear membrane. When the amino-terminal domain was deleted from LBR and replaced by alpha-globin, the chimeric protein was retained in the ER. These findings demonstrate that the amino-terminal domain of LBR is targeted to the nucleus after synthesis in the cytoplasm and that this polypeptide can function as a nuclear envelope targeting signal when located at the amino terminus of a type II integral membrane protein synthesized on the ER.

Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane.

We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxyl-terminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from approximately 22.5 to approximately 70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.

The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains.

The lamin B receptor is a previously identified integral membrane protein in the nuclear envelope of turkey erythrocytes that associates with the nuclear intermediate filament protein lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). In the present report, we use cell fractionation and antibodies against the lamin B receptor to localize it to an 8-M urea-extracted membrane fraction of chicken liver nuclei, supporting an inner nuclear membrane localization. We deduced the amino acid sequence of the chicken lamin B receptor from overlapping clones obtained by screening cDNA libraries with a probe generated by the polymerase chain reaction with primers based on the partial protein sequence of the isolated protein. The mature lamin B receptor has a calculated molecular mass of 73,375 D and eight segments of hydrophobic amino acids that could function as transmembrane domains as determined by hydropathy analysis. Preceding the first putative transmembrane segment is a highly charged 204-residue-long amino terminal region that contains two consensus sites for phosphorylation by protein kinase A. Since the lamin B receptor has been shown to be phosphorylated by protein kinase A in vitro and in vivo and this phosphorylation affects lamin B binding (Applebaum, J., G. Blobel, and S. D. Georgatos. 1990. J. Biol. Chem. 265:4181-4185), it is likely that this amino terminal region faces the nucleoplasm. The amino terminal region also contains three DNA-binding motifs that are found in gene regulatory proteins and histones, suggesting that the lamin B receptor may additionally play a role in gene regulation and/or chromatin organization.

Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis.

The mechanisms of localization and retention of membrane proteins in the inner nuclear membrane and the fate of this membrane system during mitosis were studied in living cells using the inner nuclear membrane protein, lamin B receptor, fused to green fluorescent protein (LBR-GFP). Photobleaching techniques revealed the majority of LBR-GFP to be completely immobilized in the nuclear envelope (NE) of interphase cells, suggesting a tight binding to heterochromatin and/or lamins. A subpopulation of LBR-GFP within ER membranes, by contrast, was entirely mobile and diffused rapidly and freely (D = 0. 41 +/- 0.1 microm2/s). High resolution confocal time-lapse imaging in mitotic cells revealed LBR-GFP redistributing into the interconnected ER membrane system in prometaphase, exhibiting the same high mobility and diffusion constant as observed in interphase ER membranes. LBR-GFP rapidly diffused across the cell within the membrane network defined by the ER, suggesting the integrity of the ER was maintained in mitosis, with little or no fragmentation and vesiculation. At the end of mitosis, nuclear membrane reformation coincided with immobilization of LBR-GFP in ER elements at contact sites with chromatin. LBR-GFP-containing ER membranes then wrapped around chromatin over the course of 2-3 min, quickly and efficiently compartmentalizing nuclear material. Expansion of the NE followed over the course of 30-80 min. Thus, selective changes in lateral mobility of LBR-GFP within the ER/NE membrane system form the basis for its localization to the inner nuclear membrane during interphase. Such changes, rather than vesiculation mechanisms, also underlie the redistribution of this molecule during NE disassembly and reformation in mitosis.

Structural characterization of the LEM motif common to three human inner nuclear membrane proteins.

BACKGROUND: Integral membrane proteins of the inner nuclear membrane are involved in chromatin organization and postmitotic reassembly of the nucleus. The discovery that mutations in the gene encoding emerin causes X-linked Emery-Dreifuss muscular dystrophy has enhanced interest in such proteins. A common structural domain of 50 residues, called the LEM domain, has been identified in emerin MAN1, and lamina-associated polypeptide (LAP) 2. In particular, all LAP2 isoforms share an N-terminal segment composed of such a LEM domain that is connected to a highly divergent LEM-like domain by a linker that is probably unstructured. RESULTS: We have determined the three-dimensional structures of the LEM and LEM-like domains of LAP2 using nuclear magnetic resonance and molecular modeling. Both domains adopt the same fold, mainly composed of two large parallel alpha helices. CONCLUSIONS: The structural LEM motif is found in human inner nuclear membrane proteins and in protein-protein interaction domains from bacterial multienzyme complexes. This suggests that LEM and LEM-like domains are protein-protein interaction domains. A region conserved in all LEM domains, at the surface of helix 2, could mediate interaction between LEM domains and a common protein partner.

Developmental changes in the amount and functional state of UDP-glucuronosyltransferase.

The effect of postnatal development on the activity of liver microsomal UDP-glucuronosyltransferase was determined in male Wistar rats between 25 and 200 days of age using p-nitrophenol as aglycone. Enzyme activity (measured at 1.0 mM UDP-glucuronic acid, 0.05 mM p-nitrophenol) decreased 55% between 25 and 88 days of age and was constant thereafter. Treatment of microsomes with palmitoyl-lysophosphatidylcholine, which allows for an estimation of the amount of enzyme, showed approximately a four-fold decrease in enzyme concentration during the same period. This decrease was confirmed by Western blotting of microsomes with anti-UDP-glucuronosyltransferase antiserum. The fact that a nearly four-fold decline in enzyme concentration led to only a 55% decrease in activity indicates that there was an increase in activity per molecule of UDP-glucuronosyltransferase as the concentration of enzyme decreased. Treatment of microsomes with high pressure or detergent caused a greater extent of enzyme activation in microsomes prepared from 25 than 200 day old rats, suggesting that a fraction of the enzyme in older rats was activated in untreated microsomes. Fatty acid analysis of liver microsomal lipids during postnatal development revealed changes in docosahexaenoic acid (22:6) which correlated with levels of UDP-glucuronosyltransferase activity.

The lipid fluidity of rat colonic brush-border membrane vesicles modulates Na+-H+ exchange and osmotic water permeability.

Brush-border membrane vesicles were prepared from rat colonic epithelial cells. Steady-state fluorescence polarization techniques, using the fluorophores 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid (12-AS), revealed that benzyl alcohol (25-75 mM) but not methyl alcohol (50-125 mM) significantly increased the fluidity of these vesicles. Benzyl alcohol (50 and 75 mM) but not methyl alcohol also increased amiloride-sensitive sodium-stimulated proton efflux from these vesicles at all concentrations of sodium tested (2.5-50.0 mM), as assessed by changes in the fluorescence of acridine orange. Benzyl alcohol, at 50 and 75 mM concentrations, increased the maximal velocity (Vmax) of this exchange process by approximately 58 and 75%, respectively. Neither concentration, however, altered the Km for sodium. Osmotic water flow, measured as rate constants of osmotic shrinkage of these vesicles using a stopped-flow nephelometric technique, was also increased by 75 mM benzyl alcohol but not by a similar concentration of methyl alcohol. The present data, therefore, demonstrate that the fluidity of rat colonic brush-border membranes can influence Na+-H+ exchange and osmotic water flow across these vesicles.

ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells.

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.

Structural analysis of emerin, an inner nuclear membrane protein mutated in X-linked Emery-Dreifuss muscular dystrophy.

Like Duchenne and Becker muscular dystrophies, Emery-Dreifuss muscular dystrophy (EDMD) is characterized by myopathic and cardiomyopathic abnormalities. EDMD has the particularity of being linked to mutations in nuclear proteins. The X-linked form of EDMD is caused by mutations in the emerin gene, whereas autosomal dominant EDMD is caused by mutations in the lamin A/C gene. Emerin colocalizes with lamin A/C in interphase cells, and binds in vitro to lamin A/C. Recent work suggests that lamin A/C might serve as a receptor for emerin. We have undertaken a structural analysis of emerin, and in particular of its N-terminal domain, which is comprised in the emerin segment critical for binding to lamin A/C. We show that region 2-54 of emerin adopts the LEM fold. This fold was originally described in the two N-terminal domains of another inner nuclear membrane protein called lamina-associated protein 2 (LAP2). The existence of a conserved solvent-exposed surface on the LEM domains of LAP2 and emerin is discussed, as well as the nature of a possible common target.

The expression of nuclear lamins in human liver: an immunohistochemical study.

The nuclear lamina is a meshwork of intermediate filaments adjacent to the inner nuclear membrane that in mammalian cells is predominantly composed of three proteins: lamin A, lamin B, and lamin C. Because lamin A and C (A-type lamins) expression has been shown to be lacking in several types of undifferentiated or rapidly proliferating cells, we investigated lamin expression in the human liver in conditions with hepatocellular regeneration (cirrhosis of various etiologies and macroregenerative nodules) and in hepatocellular carcinomas of various grades of differentiation. Immunohistochemical stains for A-type lamins and lamin B were performed on frozen tissue sections with the avidin-biotin complex method. Normal and regenerating hepatocytes, biliary epithelial cells (ductal and ductular cells), and hepatocellular carcinoma cells invariably expressed both A-type lamins and lamin B. These findings indicate that in hepatocellular regeneration and malignant transformation the production of both A-type lamins and lamin B is preserved.

Anti-neutrophil antibodies in primary sclerosing cholangitis.

Anti-neutrophil specific antibodies are detected in up to 88% of patients with PSC. The labelling pattern of neutrophils produced by these antibodies when examined by indirect immunofluorescence microscopy is distinct from that produced by anti-neutrophil cytoplasmic antibodies (ANCA) in vasculitic diseases. The antigen(s) recognized by anti-neutrophil antibodies in PSC is not yet known but appears to be localized to the periphery of the nucleus. Accordingly, the term peripheral anti-neutrophil nuclear antibodies (p-ANNA) is more appropriate than the frequently used p-ANCA. As the titre of p-ANNA in PSC does not correlate with disease-specific clinical parameters, they are not useful markers for the management of patients. However, the high prevalence of p-ANNA in PSC makes them a reasonable diagnostic marker if used in conjunction with other standard diagnostic tests. The role of anti-neutrophil antibodies in the pathogenesis of PSC, if any, remains to be established.

Primary biliary cirrhosis and the molecular cell biology of the nuclear envelope.

I hope I have demonstrated how basic research on the molecular cell biology of the nuclear envelope has provided information about the autoimmune disease PBC. I have given several examples of how highly specific immunologic reagents, obtained from patients with this disease, have been of value in experiments on the basic cell biology of the nuclear envelope. Continued work should provide further clues on how autoimmunity underlies the pathophysiology of PBC and should also provide additional reagents to study the processes of nuclear protein targeting and cell division.

Biliary cystadenocarcinoma arising in a liver with fibropolycystic disease.

Intrahepatic cholangiocarcinoma associated with fibropolycystic disease of the liver and biliary cystadenocarcinoma are rare tumors that are considered distinct entities. We present a case of a malignant tumor with features of hepatic cystadenocarcinoma arising in a background of fibropolycystic disease.

Escalating interferon-alpha-2b dose for patients with chronic hepatitis C.

BACKGROUND/AIMS: We examined the effectiveness of escalating the dose of interferon-alpha-2b in subjects with chronic hepatitis C who did not respond to usual treatment with 3,000,000 units 3 times a week. METHODOLOGY: Treatment was started with 3,000,000 units of interferon-alpha-2b 3 times a week. If serum alanine aminotransferase activity was not normal at 12 weeks, the dose was increased to 3,000,000 units daily. If serum alanine aminotransferase activity was not normal after 12 weeks, the dose was increased to 5,000,000 units daily. RESULTS: Fifty-one subjects started treatment. Twenty-nine subjects had their dose increased to 3,000,000 units daily and only 1 responded (3%, 95% confidence interval 0-10.9%) while 41% (95% confidence interval 21.4-60.6%) had to discontinue treatment at this dose because of adverse events or intolerance. Of 14 subjects who had their dose increased to 5,000,000 units daily, none (95% confidence interval 0-3.6%) responded, while 43% (95% confidence interval 13.5-72.5%) had to discontinue treatment. CONCLUSIONS: Escalating doses of interferon-alpha-2b are not effective and are associated with increased toxicity and intolerance in patients with chronic hepatitis who do not respond to initial treatment with 3,000,000 units 2 times a week.