Pathogenesis of the Novel Autoimmune-Associated Long-QT Syndrome.

BACKGROUND: Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)-positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go-related gene) K(+) channel, which conducts the rapidly activating delayed K(+) current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the ECG. METHODS AND RESULTS: Anti-Ro Ab-positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen-immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native IKr and cross-reacted with guinea pig ERG channel. CONCLUSIONS: The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between anti-52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

The purinergic P2×7 receptor is expressed on monocytes in Behçet's disease and is modulated by TNF-α.

The P2×7 receptor (P2×7r) is expressed in innate immune cells (e.g. monocyte/macrophages), playing a key role in IL-1ß release. Since innate immune activation and IL-1ß release seem to be implicated in Behçet's disease (BD), a systemic immune-inflammatory disorder of unknown origin, we hypothesized that P2×7r is involved in the pathogenesis of the disease. Monocytes were isolated from 18 BD patients and 17 healthy matched controls. In BD monocytes, an increased P2×7r expression and Ca(2+) permeability induced by the selective P2×7r agonist 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) was observed. Moreover, IL-1ß release from LPS-primed monocytes stimulated with BzATP was markedly higher in BD patients than in controls. TNF-α-incubated monocytes from healthy subjects almost reproduced the findings observed in BD patients, as demonstrated by the increase in P2×7r expression and BzATP-induced Ca(2+) intake. Our results provide evidence that in BD monocytes both the expression and function of the P2×7r are increased compared with healthy controls, as the possible result, at least in part, of a positive modulating effect of TNF-α on the receptor. These data indicate P2×7r as a new potential therapeutic target for the control of BD, further supporting the rationale for the use of anti-TNF-α drugs in the treatment of the disease.

Adenosine A2A receptor activation stimulates collagen production in sclerodermic dermal fibroblasts either directly and through a cross-talk with the cannabinoid system.

Systemic sclerosis (SSc) is a connective tissue disease characterised by exaggerated collagen deposition in the skin and visceral organs. Adenosine A2A receptor stimulation (A2Ar) promotes dermal fibrosis, while the cannabinoid system modulates fibrogenesis in vitro and in animal models of SSc. Moreover, evidence in central nervous system suggests that A2A and cannabinoid (CB1) receptors may physically and functionally interact. On this basis, we investigated A2Ar expression and function in modulating collagen biosynthesis from SSc dermal fibroblasts and analysed the cross-talk with cannabinoid receptors. In sclerodermic cells, A2Ar expression (RT-PCR, Western blotting) was evaluated together with the effects of A2A agonists and/or antagonists on collagen biosynthesis (EIA, Western blotting). Putative physical and functional interactions between the A2A and cannabinoid receptors were respectively assessed by co-immuno-precipitation and co-incubating the cells with the unselective cannabinoid agonist WIN55,212-2, and the selective A2A antagonist ZM-241385. In SSc fibroblasts, (1) the A2Ar is overexpressed and its occupancy with the selective agonist CGS-21680 increases collagen production, myofibroblast trans-differentiation, and ERK-1/2 phosphorylation; (2) the A2Ar forms an heteromer with the cannabinoid CB1 receptor; and (3) unselective cannabinoid receptor stimulation with a per se ineffective dose of WIN55,212-2, results in a marked anti-fibrotic effect after A2Ar blockage. In conclusion, A2Ar stimulation induces a pro-fibrotic phenotype in SSc dermal fibroblasts, either directly, and indirectly, by activating the CB1 cannabinoid receptor. These findings increase our knowledge of the pathophysiology of sclerodermic fibrosis also further suggesting a new therapeutic approach to the disease.

Proteolytic processing of the gamma-subunit is associated with the failure to form GlcNAc-1-phosphotransferase complexes and mannose 6-phosphate residues on lysosomal enzymes in human macrophages.

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

Loss of N-acetylglucosamine-1-phosphotransferase gamma subunit due to intronic mutation in GNPTG causes mucolipidosis type III gamma: Implications for molecular and cellular diagnostics.

Mucolipidosis type III gamma (MLIII, pseudo-Hurler polydystrophy) is a rare autosomal recessive disorder where the activity of the multimeric GlcNAc-1-phosphotransferase is reduced and formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes is impaired. In this disease, the targeting of lysosomal enzymes is affected resulting in their hypersecretion, and an intracellular deficiency of multiple hydrolases. We report the biochemical and molecular diagnosis of MLIII in three siblings, aged 17, 15, and 14 years, who presented with joint pain and progressive joint stiffness. In addition to missorting of newly synthesized lysosomal protease cathepsin D, there were low levels of M6P-containing proteins in cell extracts and media of cultured fibroblasts of the Patients. Direct sequencing identified a novel homozygous mutation in intron 7, IVS7-10G>A, of the GNPTG gene, which encodes the gamma-subunit of the GlcNAc-1-phosphotransferase. This mutation created a cryptic 3'-splice site resulting in a frameshift and premature translational termination (p.V176GfsX18). The GNPTG mRNA levels were markedly reduced in Patients' fibroblasts indicating that the intronic mutation mediates mRNA decay, which was confirmed by absence of the gamma-subunit protein. These data contribute to an efficient diagnostic strategy to identify Patients with MLIII gamma and characterize their biochemical defect in fibroblasts.

Compensatory expression of human N-acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma.

The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markersessential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (alpha2, beta2, gamma2). The alpha- and beta-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the gamma-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GNPTG cause mucolipidosis type III gamma, which is characterized by missorting and cellular loss of lysosomal enzymes leading to lysosomal accumulation of storage material. Using plasmon resonance spectrometry, we showed that recombinant gamma-subunit failed to bind the lysosomal enzyme arylsulfatase A. Additionally, the overexpression of the gamma-subunit in COS7 cells did not result in hypersecretion of newly synthesized lysosomal enzymes expected for competition for binding sites of the endogenous phosphotransferase complex. Analysis of fibroblasts exhibiting a novel mutation in GNPTG (c.619insT, p.K207IfsX7) revealed that the expression of GNPTAB was increased whereas in gamma-subunit overexpressing cells the GNPTAB mRNA was reduced. The data suggest that the gamma-subunit is important for the balance of phosphotransferase subunits rather for general binding of lysosomal enzymes.